Chimeric cytokine receptor

ABSTRACT

The present invention provides a chimeric cytokine receptor (CCR) comprising: (i) an exodomain which binds to a ligand selected from a tumour secreted factor, a chemokine and a cell-surface antigen; and (ii) a cytokine receptor endodomain.

FIELD OF THE INVENTION

The present invention relates to a chimeric cytokine receptor (CCR), anda cell which expresses such a chimeric cytokine receptor and optionallya chimeric antigen receptor at the cell surface.

BACKGROUND TO THE INVENTION

Chimeric Antigen Receptors (CARs)

A number of immunotherapeutic agents have been described for use incancer treatment, including therapeutic monoclonal antibodies (mAbs),bi-specific T-cell engagers and chimeric antigen receptors (CARs).

Chimeric antigen receptors are proteins which graft the specificity of amonoclonal antibody (mAb) to the effector function of a T-cell. Theirusual form is that of a type I transmembrane domain protein with anantigen recognizing amino terminus, a spacer, a transmembrane domain allconnected to a compound endodomain which transmits T-cell survival andactivation signals.

The most common form of these molecules are fusions of single-chainvariable fragments (scFv) derived from monoclonal antibodies whichrecognize a target antigen, fused via a spacer and a trans-membranedomain to a signaling endodomain. Such molecules result in activation ofthe T-cell in response to recognition by the scFv of its target. When Tcells express such a CAR, they recognize and kill target cells thatexpress the target antigen. Several CARs have been developed againsttumour associated antigens, and adoptive transfer approaches using suchCAR-expressing T cells are currently in clinical trial for the treatmentof various cancers.

CAR-Based Approaches to Treat Prostate Cancer

Prostate cancer is the second most common cancer in men worldwide, andthe sixth leading cause of cancer-related death. Globally, there areapproximately 1,100,000 new cases and 300,000 mortalities every year,comprising 4 percent of all cancer deaths. It is estimated that 1 inevery 6 men will be diagnosed with the disease during his lifetime.

Initial treatment for prostate cancer may consist of surgery, radiation,or hormone therapy, or any combination of each. Hormone therapy consistsof lowering the levels of testosterone, the male hormone that fuelsout-of-control cell growth. Chemotherapy is typically reserved foradvanced-stage cancers.

When prostate cancers grow despite the lowering of testosterone levelsby hormone therapy, treatment options are limited. Typically, the cancervaccine sipuleucel-T (Provenge®) a dendritic cell-based therapeuticcancer vaccine designed to induce an immune response targeted againstthe prostatic acid phosphatase ((PAP) antigen), a radiopharmaceuticalagent (such as radium-223 chloride), secondary hormone therapies (suchas abiraterone or enzalutamide), and/or chemotherapies (docetaxel andcabazitaxel) are added to the hormonal therapy in sequence. While eachof these treatments can delay growth of the cancer for several monthsand palliate symptoms produced by the disease, the disease ultimatelybecomes resistant to them.

Preclinically, two antigens associated with prostate cancer have beentargeted with CAR T-cell based therapies: prostate-specific membraneantigen (PSMA) and prostate stem cell antigen (PSCA).

Mice treated with PSCA CAR-engineered T cells showed delayed tumourgrowth (Hillerdal et al (2014) BMC Cancer 14:30; and Abate-Daga et al(2014) 25:1003-1012). Although the cells showed high in vitrocytotoxicity, in vivo, tumour growth was delayed but tumour-bearing micewere not cured.

This may be because, in vivo, CAR T-cells struggle to overcome thehostile microenvironment of a carcinoma. In particular CAR T-cells mayfail to engraft and expand within a prostate cancer tumour bed.

CAR T-cell persistence and activity can be enhanced by administration ofcytokines, or by the CAR T-cells producing cytokines constitutively.However, these approaches have limitations: systemic administration ofcytokines can be toxic; constitutive production of cytokines may lead touncontrolled proliferation and transformation (Nagarkatti et al (1994)PNAS 91:7638-7642; Hassuneh et al (1997) Blood 89:610-620).

There is therefore a need for alternative CAR T-cell approaches, whichfacilitate engraftment and expansion of T cells to counteract theeffects of the hostile tumour microenvironment.

On-Target Off-Tumour Toxicity

It is relatively rare for the presence of a single antigen effectivelyto describe a cancer, which can lead to a lack of specificity.

Most cancers cannot be differentiated from normal tissues on the basisof a single antigen. Hence, considerable “on-target off-tumour” toxicityoccurs whereby normal tissues are damaged by the therapy. For instance,whilst targeting CD20 to treat B-cell lymphomas with Rituximab, theentire normal B-cell compartment is depleted, whilst targeting CD52 totreat chronic lymphocytic leukaemia, the entire lymphoid compartment isdepleted, whilst targeting CD33 to treat acute myeloid leukaemia, theentire myeloid compartment is damaged etc.

The predicted problem of “on-target off-tumour” toxicity has been borneout by clinical trials. For example, an approach targeting ERBB2 causeddeath to a patient with colon cancer metastatic to the lungs and liver.ERBB2 is over-expressed in colon cancer in some patients, but it is alsoexpressed on several normal tissues, including heart and normalvasculature.

There is therefore a need for improved approaches to cancer therapy inwhich such “on-target off-tumour” toxicity is reduced or eliminated.

DESCRIPTION OF THE FIGURES

FIG. 1: Schematic diagram summarising the structure of various cytokinereceptors, the cell types which produce the cytokines and the cell typeswhich express the cytokine receptors.

FIG. 2: Schematic diagram showing proposed chimeric cytokine receptor

(a) Cytokine IL2 and IL7 cytokine receptors signal through a commongamma chain and a cytokine specific alpha/beta chain.(b) One implementation of a chimeric cytokine receptor is to replace theectodomain of the cytokine alpha/beta and gamma chain with differentscFvs (or any other suitable binder) which recognize different epitopesof PSA.(c) An alternative approach is to replace the ectodomains of alpha/betaand gamma with the VH/VL of a PSA specific antibody, where both VH andVL are involved in binding so that binding brings them together.

FIG. 3: Aggregation-based cytokine signalling enhancer

Schematic diagram showing a chimeric cytokine receptor and CARcombination system. The cell comprises two chimeric cytokine receptorswhich bind different epitopes on the same soluble ligand. In the absenceof soluble ligand (e.g. PSA) but the presence of the cell-membraneantigen (e.g. PSMA) signalling occurs thought the CAR. In the presenceof the soluble ligand, aggregation of the two chimeric cytokinereceptors occurs, leading to cytokine-based signal enhancement.

FIG. 4: Theoretical construct map for the chimeric cytokine receptor/CARcombination system illustrated in FIG. 3.

FIG. 5: Schematic diagram illustrating an example of a structure for thechimeric transmembrane protein of the present invention. The chimerictransmembrane protein comprises a dimerization domain and a cytokinereceptor endodomain. The embodiment shown has a “Fab” type architecture,as the dimerization domain comprises antibody-type heavy and light chainconstant regions. Constant dimerization between these domains bringstogether the IL2 receptor common γ chain with either the IL-2 receptor βchain or the IL-7 receptor α chain, leading to constitutive cytokinesignalling.

FIG. 6: IL-2 signalling by the chimeric transmembrane protein.

Two chimeric transmembrane proteins having the general structure shownin FIG. 5 were tested for their ability to induce IL-2 signalling. Onechimeric transmembrane protein comprised an IL2 receptor endodomain andthe other comprised an IL-7 receptor endodomain. IL-2 signalling wastested using the murine cell line CTLL2 which is dependent on IL-2signalling for growth. As a positive control, CTLL2 cells were culturedwith 100 u/mL murine IL2. Cells expressing the chimeric transmembraneprotein comprising the IL2 receptor endodomain (Fab_IL2endo) supportedCTLL2 cell survival and growth, whereas cells expressing the chimerictransmembrane protein comprising the IL-7 receptor (Fab_IL7endo) didnot.

FIG. 7: Schematic diagram illustrating panel of PSA chimeric cytokinereceptors A panel of chimeric cytokine receptors (CCRs) targeting PSAwas developed using scFvs derived from two antibodies which bind todifferent PSA epitopes: 5D5A5 and 5D3D11.

Top-left panel: A CCR with an IL-2R endodomain having A5 on the chainwith IL2R chain and D11 on the chain with common γ chain;

Top-right panel: A CCR with an IL7R endodomain having A5 on the chainwith IL7R α chain and D11 on the chain with common γ chain;

Bottom-left panel: A CCR with an IL-2R endodomain having D11 on thechain with IL2R 13 chain and A5 on the chain with common γ chain; and

Bottom-right hand panel: A CCR with an IL-7R endodomain having D11 onthe chain with IL7R α chain and A5 on the chain with common γ chain.

A negative control was also created for each CCR, in which the IL2Rγchain was replaced by a rigid linker.

FIG. 8: IL2 signalling from cells expressing a PSA chimeric cytokinereceptor in the presence of PSA-CTLL2 proliferation

CTLL2 cells were transduced with constructs expressing some of the PSAchimeric cytokine receptors illustrated in FIG. 7. Cells were culturedin the presence of absence of IL2 (positive control) and the presence ofabsence of 5 ng/mL or 5 μg/mL PSA. CTLL2 proliferation was assessedafter 3 and 7 days.

The anti-PSA chimeric cytokine receptor with an IL2R endodomainsupported CTLL2 cell proliferation in the absence of IL2 and thepresence of PSA, but not the receptor having an IL7R endodomain or anyof the CCRs comprising a rigid linker in the place of the common γchain.

FIG. 9: IL2 signalling from cells expressing a PSA chimeric cytokinereceptor in the presence of PSA-CTLL2 STAT5 phosphorylation

CTLL2 cells were either left untransduced (VVT); or transduced with avector expressing a CCR against PSA (D11-CD8STK-IL2Rg_A5-Hinge-IL2Rb) oran equivalent construct having a rigid linker in the place of the commonγ chain (D11-CD8STK-RL_A5-Hinge-IL2Rb). Cells were incubated with either500 μM Pervanadate or 500 ng/mL PSA for 1 or 4 hours. Phosphorylation ofY694 of STAT5 was then investigated using phosphoflow.

SUMMARY OF ASPECTS OF THE INVENTION

The present inventors have developed “chimeric cytokine receptors” (CCR)which graft the binding specificity of a non-cytokine binding moleculeon to the endodomain of a cytokine receptor. The co-expression of such aCCR with a chimeric antigen receptor (CAR) helps a CAR T-cell to engraftand expand in the hostile tumour microenvironment. The requirement forthe ligand for the CCR as well as the ligand for the CAR to be presentadd another layer of selectivity and helps prevent on-target off-tumourtoxicity.

For example, they have developed a cell which co-expresses a CAR with achimeric cytokine receptor which detects PSA and transmits an IL2/15 oran IL7 signal to the CAR T-cell. In this way, the CAR T-cell isstimulated to proliferate selectively only in a prostate cancermicroenvironment, and in the absence of PSA (i.e. after the patient isin remission), the cytokine stimulation is lost.

In a first aspect, the present invention provides a chimeric cytokinereceptor (CCR) comprising:

-   -   an exodomain which binds to a ligand selected from a tumour        secreted factor, a chemokine and a cell-surface antigen; and    -   a cytokine receptor endodomain.

In a first embodiment of the first aspect of the invention, the chimericcytokine receptor comprises two polypeptides:

-   -   (i) a first polypeptide which comprises:        -   (a) a first antigen-binding domain which binds a first            epitope of the ligand        -   (b) a first chain of the cytokine receptor endodomain; and    -   (ii) a second polypeptide which comprises:        -   (a) a second antigen-binding domain which binds a second            epitope of the ligand        -   (b) a second chain of the cytokine-receptor endodomain. FIG.            2b illustrates such an arrangement.

Each of the first and second antigen-binding domains may be, forexample, single-chain variable fragments (scFvs) or single domainbinders.

In a second embodiment of the first aspect of the invention, thechimeric cytokine receptor which comprises two polypeptides:

-   -   (i) a first polypeptide which comprises:        -   (a) a heavy chain variable domain (VH)        -   (b) a first chain of the cytokine receptor endodomain; and    -   (ii) a second polypeptide which comprises:        -   (a) a light chain variable domain (VL)        -   (b) a second chain of the cytokine-receptor endodomain.

FIG. 2c illustrates such an arrangement.

The first and second chains for the cytokine receptor endodomains may bedifferent and may be selected from type I cytokine receptor endodomainα-, β-, and γ-chains.

Alternatively the first and second chains for the cytokine receptorendodomains may be the same and may be selected from type I cytokinereceptor endodomain α-, β-, and γ-chains.

For example, the cytokine receptor endodomain may comprise:

-   -   (i) IL-2 receptor β-chain endodomain    -   (ii) IL-7 receptor α-chain endodomain;    -   (iii) IL-15 receptor α-chain endodomain; or    -   (iv) common γ-chain receptor endodomain.

The cytokine receptor endodomain may comprise (i), (ii) or (iii); and(iv).

The ligand may be a tumour secreted factor, for example a tumoursecreted factor selected from: prostate-specific antigen (PSA),carcinoembryonic antigen (CEA), vascular endothelial growth factor(VEGF) and CA125.

The ligand may be a chemokine, for example a chemokine selected fromchemokine selected from: CXCL12, CCL2, CCL4, CCL5 and CCL22.

The ligand may be a cell-surface molecule, such as a transmembraneprotein. The ligand may be, for example, CD22.

In a second aspect, the present invention provides a cell whichcomprises a chimeric cytokine receptor according to the first aspect ofthe invention.

The cell may comprise a first chimeric cytokine receptor and a secondchimeric cytokine receptor which bind different epitopes on the sameligand.

The cell may comprise a first chimeric cytokine receptor which comprisesa type I cytokine receptor endodomain α- or β-chain, and a secondchimeric cytokine receptor which comprises a type I cytokine receptorendodomain γ-chain, such that when the first chimeric cytokine receptorand the second cytokine receptor bind the ligand, combined signallingthrough the α-/β-chain and γ-chain occurs.

The cell may also comprise a chimeric antigen receptor, for example achimeric antigen receptor which binds a tumour-associated cell surfaceantigen.

The chimeric antigen receptor may bind a cell surface antigen associatedwith prostate cancer, such as prostate stem-cell antigen (PSCA) orprostate-specific membrane antigen (PSMA).

Where the CCR recognises a cell-surface antigen, the CCR and CAR mayrecognise cell-surface antigens which are co-expressed on the sametarget (e.g. tumour) cell. For example, for B-cell malignancies, the CARmay recognize a cell-surface antigen such as CD19 and the CCR mayrecognize a molecule which is co-expressed on the target cell surface,such CD22, thereby enhancing engraftment.

In a third aspect, the present invention provides a nucleic acidsequence encoding a chimeric cytokine receptor (CCR) according to thefirst aspect of the invention.

In a fourth aspect the present invention provides a nucleic acidconstruct which comprises a first nucleic acid sequence encoding a firstCCR and a second nucleic acid sequence encoding a second CCR, thenucleic acid construct having the structure:

-   -   AgB1-spacer1-TM1-endo1-coexpr-AbB2-spacer2-TM2-endo2 in which    -   AgB1 is a nucleic acid sequence encoding the antigen-binding        domain of the first CCR;    -   spacer 1 is a nucleic acid sequence encoding the spacer of the        first CCR;    -   TM1 is a a nucleic acid sequence encoding the transmembrane        domain of the first CCR;    -   endo 1 is a nucleic acid sequence encoding the endodomain of the        first CCR;    -   coexpr is a nucleic acid sequence enabling co-expression of both        CCRs    -   AgB2 is a nucleic acid sequence encoding the antigen-binding        domain of the second CCR;    -   spacer 2 is a nucleic acid sequence encoding the spacer of the        second CCR;    -   TM2 is a a nucleic acid sequence encoding the transmembrane        domain of the second CCR;    -   endo 2 is a nucleic acid sequence encoding the endodomain of the        second CCR.

The nucleic acid construct may also encode a chimeric antigen receptor(CAR). In this embodiment, the nucleic acid construct may have thestructure:

-   -   (i)        CCRAgB1-CCRspacer1-CCRTM1-CCRendo1-coexpr1-CCRAgB2-CCRspacer2-CCRTM2-CCRendo2-coexpr2-CARAgB-CARspacer-CARTM-CARendo;    -   (ii)        CCRAgB1-CCRspacer1-CCRTM1-CCRendo1-coexpr1-CARAgB-CARspacer-CARTM-CARendo-coexpr2-CCRAgB2-CCRspacer2-CCRTM2-CCRendo2;        or    -   (iii)        CARAgB-CARspacer-CARTM-CARendo-coexpr1-CCRAgB1-CCRspacer1-CCRTM1-CCRendo1-coexpr2-CCRAgB2-CCRspacer2-CCRTM2-CCRendo2;    -   in which    -   CCRAgB1 is a nucleic acid sequence encoding the antigen-binding        domain of the first CCR;    -   CCRspacer1 is a nucleic acid sequence encoding the spacer of the        first CCR;    -   CCRTM1 is a nucleic acid sequence encoding the transmembrane        domain of the first CCR;    -   CCRendo1 is a nucleic acid sequence encoding the endodomain of        the first CCR;    -   CCRAgB2 is a nucleic acid sequence encoding the antigen-binding        domain of the second CCR;    -   CCRspacer2 is a nucleic acid sequence encoding the spacer of the        second CCR;    -   CCRTM2 is a nucleic acid sequence encoding the transmembrane        domain of the second CCR;    -   CCRendo2 is a nucleic acid sequence encoding the endodomain of        the second CCR;    -   Coexpr1 and coexpr2 are nucleic acid sequences enabling        co-expression of the two flanking sequences;    -   CARAgB is a nucleic acid sequence encoding the antigen-binding        domain of the CAR;    -   CARspacer is a nucleic acid sequence encoding the spacer of the        CAR;    -   CARTM is a nucleic acid sequence encoding the transmembrane        domain of the CAR; and    -   CARendo is a nucleic acid sequence encoding the endodomain of        the CAR.

Any or all of the sequences coexpr, coexpr1, coexpr2 may encode asequence comprising a self-cleaving peptide.

Alternative codons may be used in regions of sequence encoding the sameor similar amino acid sequences, in order to avoid homologousrecombination.

In a fifth aspect, the present invention provides a vector comprising anucleic acid construct according to the fourth aspect of the invention.

The vector may be, for example, a retroviral vector or a lentiviralvector or a transposon.

In a sixth aspect, the present invention provides a kit which comprises:

-   -   i) a vector comprising a nucleic acid sequence encoding a first        CCR according to the first aspect of the invention; and    -   ii) a vector comprising a nucleic acid sequence encoding a        second CCR according to the second aspect of the invention.

The kit may also comprise a vector comprising a nucleic acid sequenceencoding a chimeric antigen receptor.

The kit may comprise:

-   -   i) a vector comprising a nucleic acid sequence encoding a CCR        according to the first aspect of the invention; and    -   ii) a vector comprising a nucleic acid sequence encoding a        chimeric antigen receptor.

In a seventh aspect, the present invention provides a method for makinga cell according to the second aspect of the invention, which comprisesthe step of introducing: a nucleic acid sequence according to the thirdaspect of the invention; a nucleic acid construct according to thefourth aspect of the invention; a vector according to the fifth aspectof the invention; or a kit of vectors according to the sixth aspect ofthe invention, into a cell.

The cell may be from a sample isolated from a subject.

In an eighth aspect, there is provided a pharmaceutical compositioncomprising a plurality of cells according to the second aspect of theinvention.

In a ninth aspect, there is provided a method for treating and/orpreventing a disease, which comprises the step of administering apharmaceutical composition according to the eighth aspect of theinvention to a subject.

The method may comprise the following steps:

-   -   (i) isolation of a cell-containing sample from a subject;    -   (ii) transduction or transfection of the cells with: a nucleic        acid sequence according to the third aspect of the invention; a        nucleic acid construct according to the fourth aspect of the        invention; a vector according to the fifth aspect of the        invention; or a kit of vectors according to the sixth aspect of        the invention; and    -   (iii) administering the cells from (ii) to a the subject.

The sample may be a T-cell containing sample.

The disease may be a cancer.

There is also provided a pharmaceutical composition according to theeighth aspect of the invention for use in treating and/or preventing adisease.

There is also provided the use of a cell according to the second aspectof the invention in the manufacture of a medicament for treating and/orpreventing a disease.

Further aspects of the invention are summarised in the followingnumbered paragraphs:

-   -   1. A chimeric transmembrane protein comprising:    -   a dimerization domain; and    -   a cytokine receptor endodomain.    -   2. A chimeric transmembrane protein according to paragraph 1,        wherein the dimerization domain comprises the dimerization        portion of a heavy chain constant domain (C_(H)) and a light        chain constant domain (C_(L)).    -   3. A chimeric transmembrane protein according to any preceding        paragraph, which comprises two polypeptides:        -   (i) a first polypeptide which comprises:            -   (a) a first dimerisation domain; and            -   (b) a first chain of the cytokine receptor endodomain;                and        -   (ii) a second polypeptide which comprises:            -   (a) a second dimerization domain, which dimerises with                the first dimerization domain; and            -   (b) a second chain of the cytokine-receptor endodomain.    -   4. A chimeric transmembrane protein according to paragraph 3,        wherein the first and second dimerization domains either        dimerise spontaneously, or in the presence of a chemical inducer        of dimerization (CID).    -   5. A chimeric transmembrane protein according to paragraph 2, 3        or 4 which comprises two polypeptides:        -   (i) a first polypeptide which comprises:            -   (a) a heavy chain constant domain (CH)            -   (b) a first chain of the cytokine receptor endodomain;                and        -   (ii) a second polypeptide which comprises:            -   (a) a light chain constant domain (CL)            -   (b) a second chain of the cytokine-receptor endodomain.    -   6. A chimeric transmembrane protein according to paragraph 5        wherein the first and second chains for the cytokine receptor        endodomains are different and are selected from type I cytokine        receptor endodomain α-, β-, and γ-chains.    -   7. A chimeric transmembrane protein according to paragraph 5        wherein the first and second chains for the cytokine receptor        endodomains are the same and are selected from type I cytokine        receptor endodomain α-, β-, and γ-chains.    -   8. A chimeric transmembrane protein according to any preceding        paragraph, wherein the cytokine receptor endodomain comprises:    -   (i) IL-2 receptor β-chain endodomain    -   (ii) IL-7 receptor α-chain endodomain; or    -   (iii) IL-15 receptor α-chain endodomain; and/or    -   (iv) common γ-chain receptor endodomain.    -   9. A chimeric transmembrane protein according to paragraph 5,        wherein the first polypeptide comprises a heavy chain variable        domain (VH) and a heavy chain constant domain (CH); and the        second polypeptide comprises a light chain variable domain (VL)        and a light chain constant domain (CL).    -   10. A chimeric transmembrane protein according to paragraph 9        which comprises a Fab exodomain.    -   11. A cell which comprises a chimeric transmembrane protein        according to any preceding paragraph.    -   12. A cell according to paragraph 11, which also comprises a        chimeric antigen receptor.    -   13. A cell according to paragraph 12, wherein the chimeric        antigen receptor binds a tumour-associated cell surface antigen.    -   14. A nucleic acid sequence encoding a chimeric transmembrane        protein according to any of paragraphs 1 to 10.    -   15. A nucleic acid construct which comprises a first nucleic        acid sequence encoding a first polypeptide as defined in        paragraph 3 and a second nucleic acid sequence encoding a second        polypeptide as defined in paragraph 3, the nucleic acid        construct having the structure:    -   Dim1-TM 1-endo1-coexpr-Dim2-TM2-endo2    -   in which    -   Dim1 is a nucleic acid sequence encoding the first dimerisation        domain;    -   TM1 is a a nucleic acid sequence encoding the transmembrane        domain of the first polypeptide;    -   endo 1 is a nucleic acid sequence encoding the endodomain of the        first polypeptide;    -   coexpr is a nucleic acid sequence enabling co-expression of both        CCRs    -   Dim2 is a nucleic acid sequence encoding the second dimerization        domain;    -   TM2 is a a nucleic acid sequence encoding the transmembrane        domain of the second polypeptide;    -   endo 2 is a nucleic acid sequence encoding the endodomain of the        second polypeptide.    -   16. A nucleic acid construct according to paragraph 15 which        also encodes a chimeric antigen receptor (CAR).    -   17. A nucleic acid construct according to paragraph 15 or 16,        wherein coexpr encodes a sequence comprising a self-cleaving        peptide.    -   18. A nucleic acid construct according to any of paragraphs 15        to 17, wherein alternative codons are used in regions of        sequence encoding the same or similar amino acid sequences, in        order to avoid homologous recombination.    -   19. A vector comprising a nucleic acid construct according to        any of paragraphs 15 to 18.    -   20. A retroviral vector or a lentiviral vector or a transposon        according to paragraph 19.    -   21. A kit which comprises:        -   i) a vector comprising a nucleic acid sequence encoding a            first polypeptide as defined in paragraph 3; and        -   ii) a vector comprising a nucleic acid sequence encoding a            second polypeptide as defined in paragraph 3.    -   22. A kit according to paragraph 21 which also comprises a        vector comprising a nucleic acid sequence encoding a chimeric        antigen receptor.    -   23. A kit which comprises:        -   i) a vector comprising a nucleic acid sequence encoding a            chimeric transmembrane protein as defined in any of            paragraphs 1 to 10; and        -   ii) a vector comprising a nucleic acid sequence encoding a            chimeric antigen receptor.    -   24. A method for making a cell according to any of paragraphs 11        to 13, which comprises the step of introducing: a nucleic acid        sequence according to paragraph 14; a nucleic acid construct        according to any of paragraphs 15 to 18; a vector according to        paragraph 19 or 20; or a kit of vectors according to any        paragraphs 21 to 23, into a cell.    -   25. A method according to paragraph 24, wherein the cell is from        a sample isolated from a subject.    -   26. A pharmaceutical composition comprising a plurality of cells        according to any of paragraphs 11 to 13.    -   27. A method for treating and/or preventing a disease, which        comprises the step of administering a pharmaceutical composition        according to paragraph 26 to a subject.    -   28. A method according to claim 27, which comprises the        following steps:        -   (i) isolation of a cell-containing sample from a subject;        -   (ii) transduction or transfection of the cells with: a            nucleic acid sequence according to paragraph 14; a nucleic            acid construct according to any of paragraphs 15 to 18; a            vector according to paragraph 19 or 20; or a kit of vectors            according to any paragraphs 21 to 23; and        -   (iii) administering the cells from (ii) to a the subject.    -   29. A method according to paragraph 28, wherein the sample is a        T-cell containing sample.    -   30. A method according to paragraph 28 or 29, wherein the        disease is a cancer.    -   31. A pharmaceutical composition according to paragraph 26 for        use in treating and/or preventing a disease.    -   32. The use of a cell according to any of paragraphs 11 to 13 in        the manufacture of a medicament for treating and/or preventing a        disease.

DETAILED DESCRIPTION Chimeric Cytokine Receptor (CCR)

A chimeric cytokine receptor (CCR) is a molecule which comprises acytokine receptor endodomain and a heterologous ligand-bindingexodomain. The heterologous exodomain binds a ligand other than thecytokine for which the cytokine receptor from which the endodomain wasderived is selective. In this way, it is possible to alter the ligandspecificity of a cytokine receptor by grafting on a heterologous bindingspecificity.

A chimeric cytokine receptor comprises:

(i) a ligand binding exodomain;

(ii) an optional spacer;

(iii) a transmembrane domain; and

(iv) a cytokine-receptor endodomain.

Cytokine Receptors and Signalling

Many cell functions are regulated by members of the cytokine receptorsuperfamily. Signalling by these receptors depends upon theirassociation with Janus kinases (JAKs), which couple ligand binding totyrosine phosphorylation of signalling proteins recruited to thereceptor complex. Among these are the signal transducers and activatorsof transcription (STATs), a family of transcription factors thatcontribute to the diversity of cytokine responses.

When the chimeric cytokine receptor of the invention binds its ligand,one or more of the following intracellular signaling pathways may beinitiated:

(i) the JAK-STAT pathway

(ii) the MAP kinase pathway; and

(iii) the Phosphoinositide 3-kinase (PI3K) pathway.

The JAK-STAT system consists of three main components: (1) a receptor(2) Janus kinase (JAK) and (3) Signal Transducer and Activator ofTranscription (STAT).

JAKs, which have tyrosine kinase activity, bind to cell surface cytokinereceptors. The binding of the ligand to the receptor triggers activationof JAKs. With increased kinase activity, they phosphorylate tyrosineresidues on the receptor and create sites for interaction with proteinsthat contain phosphotyrosine-binding SH2 domains. STATs possessing SH2domains capable of binding these phosphotyrosine residues are recruitedto the receptors, and are themselves tyrosine-phosphorylated by JAKs.These phosphotyrosines then act as binding sites for SH2 domains ofother STATs, mediating their dimerization. Different STATs form hetero-or homodimers. Activated STAT dimers accumulate in the cell nucleus andactivate transcription of their target genes.

Cytokine Receptor Endodomain

The chimeric cytokine receptor of the present invention comprises anendodomain which causes “cytokine-type” cell signalling (either alone orwhen in the presence of another chimeric cytokine receptor) when theexodomain binds its ligand.

The endodomain may be a cytokine receptor endodomain.

The endodomain may be derived from a type I cytokine receptor. Type Icytokine receptors share a common amino acid motif (WSXWS) in theextracellular portion adjacent to the cell membrane.

The endodomain may be derived from a type II cytokine receptor. Type IIcytokine receptors include those that bind type I and type IIinterferons, and those that bind members of the interleukin-10 family(interleukin-10, interleukin-20 and interleukin-22).

Type I cytokine receptors include:

-   -   (i) Interleukin receptors, such as the receptors for IL-2, IL-3,        IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-12, IL13, IL-15, IL-21,        IL-23 and IL-27;    -   (ii) Colony stimulating factor receptors, such as the receptors        for erythropoietin, GM-CSF, and G-CSF; and    -   (iii) Hormone receptor/neuropeptide receptor, such as hormone        receptor and prolactin receptor

Members of the type I cytokine receptor family comprise differentchains, some of which are involved in ligand/cytokine interaction andothers that are involved in signal transduction. For example the IL-2receptor comprises an α-chain, a β-chain and a γ-chain.

The IL-2 receptor common gamma chain (also known as CD132) is sharedbetween the IL-2 receptor, IL-4 receptor, IL-7 receptor, IL-9 receptor,IL-13 receptor and IL-15 receptor.

IL-2

IL-2 binds to the IL-2 receptor, which has three forms, generated bydifferent combinations of three different proteins, often referred to as“chains”: α, β and γ; these subunits are also parts of receptors forother cytokines. The β and γ chains of the IL-2R are members of the typeI cytokine receptor family.

The three receptor chains are expressed separately and differently onvarious cell types and can assemble in different combinations and ordersto generate low, intermediate, and high affinity IL-2 receptors.

The α chain binds IL-2 with low affinity, the combination of β and γtogether form a complex that binds IL-2 with intermediate affinity,primarily on memory T cells and NK cells; and all three receptor chainsform a complex that binds IL-2 with high affinity (Kd˜10-11 M) onactivated T cells and regulatory T cells.

The three IL-2 receptor chains span the cell membrane and extend intothe cell, thereby delivering biochemical signals to the cell interior.The alpha chain does not participate in signalling, but the beta chainis complexed with the tyrosine phosphatase JAK1. Similarly the gammachain complexes with another tyrosine kinase called JAK3. These enzymesare activated by IL-2 binding to the external domains of the IL-2R.

IL-2 signalling promotes the differentiation of T cells into effector Tcells and into memory T cells when the initial T cells are alsostimulated by an antigen. Through their role in the development of Tcell immunologic memory, which depends upon the expansion of the numberand function of antigen-selected T cell clones, they also have a keyrole in long-term cell-mediated immunity.

The chimeric cytokine receptor of the present invention may comprise theIL-2 receptor β-chain and/or the IL-2 receptor (i.e. common) γ-chain

The amino acid sequences for the endodomains of the IL-2 β-chain andcommon γ-chain are shown as SEQ ID No. 1 and 2

SEQ ID No. 1:  Endodomain derived from human common gamma chain:ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPETSEQ ID No. 2: Endodomain derived from human IL-2Rβ:NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV

The term “derived from” means that the endodomain of the chimericcytokine receptor of the invention has the same sequence as thewild-type sequence of the endogenous molecule, or a variant thereofwhich retains the ability to form a complex with JAK-1 or JAK-3 andactivate one of the signalling pathways mentioned above.

A “variant” sequence having at least 80, 85, 90, 95, 98 or 99% sequenceidentity to the wild-type sequence (e.g. SEQ ID Nos. 1 or 2), providingthat the variant sequence retains the function of the wild-type sequencei.e. the ability to form a complex with JAK-1 or JAK-3 and activate, forexample, the JAK-STAT signalling pathway.

The percentage identity between two polypeptide sequences may be readilydetermined by programs such as BLAST which is freely available athttp://blast.ncbi.nlm.nih.gov

IL-7

The interleukin-7 receptor is made up of two chains: the interleukin-7receptor-α chain (CD127) and common-γ chain receptor (CD132). Thecommon-γ chain receptors is shared with various cytokines, includinginterleukin-2, -4, -9, and -15. Interleukin-7 receptor is expressed onvarious cell types, including naive and memory T cells.

The interleukin-7 receptor plays a critical role in the development oflymphocytes, especially in V(D)J recombination. IL-7R also controls theaccessibility of a region of the genome that contains the T-cellreceptor gamma gene, by STATS and histone acetylation. Knockout studiesin mice suggest that blocking apoptosis is an essential function of thisprotein during differentiation and activation of T lymphocytes.

The chimeric cytokine receptor of the present invention may comprise theIL-7 receptor α-chain and/or the IL-7 receptor (i.e. common) γ-chain, ora variant thereof.

The amino acid sequence for the endodomain of the IL-7 α-chain is shownas SEQ ID No. 3.

SEQ ID No. 3- Endodomain derived from human IL-7Ra:KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ

IL-15

Interleukin 15 (IL-15) is a cytokine with structural similarity to IL-2.Like IL-2, IL-15 binds to and signals through a complex composed ofIL-2/1L-15 receptor beta chain (CD122) and the common gamma chain(gamma-C, CD132). IL-15 is secreted by mononuclear phagocytes (and someother cells) following viral infection. IL-15 induces cell proliferationof natural killer cells.

Interleukin-15 receptor consists of an interleukin 15 receptor alphasubunit and shares common beta and gamma subunits with the IL-2receptor.

Spacer

The chimeric cytokine receptor of the present invention may comprise aspacer to connect the antigen-binding domain with the transmembranedomain and spatially separate the antigen-binding domain from theendodomain. A flexible spacer allows to the antigen-binding domain toorient in different directions to enable antigen binding.

Where the cell of the present invention comprises two or more chimericcytokine receptors, the spacers may be the same or different. Where thecell of the present invention comprises a chimeric cytokine receptor(CCR) and a chimeric antigen receptor (CAR), the spacer of the CCR andthe CAR may be different, for example, having a different length. Thespacer of the CAR may be longer than the spacer of the or each CCR.

The spacer sequence may, for example, comprise an IgG1 Fc region, anIgG1 hinge or a CD8 stalk. The linker may alternatively comprise analternative linker sequence which has similar length and/or domainspacing properties as an IgG1 Fc region, an IgG1 hinge or a CD8 stalk.

A human IgG1 spacer may be altered to remove Fc binding motifs.

Examples of amino acid sequences for these spacers are given below:

SEQ ID No. 4 (hinge-CH2CH3 of human IgG1)AEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKD SEQ ID No. 5 (human CD8 stalk):TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDISEQ ID No. 6 (human IgG1 hinge): AEPKSPDKTHTCPPCPKDPK

Transmembrane Domain

The transmembrane domain is the sequence of a CCR that spans themembrane. It may comprise a hydrophobic alpha helix. The transmembranedomain may be derived from CD28, which gives good receptor stability.

Alternatively the transmembrane domain may be derived from a cytokinereceptor, for example the same cytokine from which the endodomain isderived.

The transmembrane domain may, for example be derived from IL-2R, IL-7Ror IL-15R.

SEQ ID No. 7 Transmembrane derived  from human common gamma chain:VVISVGSMGLIISLLCVYFWL SEQ ID No. 8 Transmembrane derived from human IL-2Rβ: IPWLGHLLVGLSGAFGFIILVYLLISEQ ID No. 9 Transmembrane derived  from human IL-7Rα:PILLTISILSFFSVALLVILACVLW SEQ ID No. 10 Transmembrane derived from human IL-15Rα: AISTSTVLLCGLSAVSLLACYL

Ligand-Binding Exodomain

The ligand binding domain comprises an antigen binding domain. Theantigen binding domain binds the target ligand for the CCR, i.e. thetumour secreted factor or chemokine or cell surface antigen.

Numerous antigen-binding domains are known in the art, including thosebased on the antigen binding site of an antibody, antibody mimetics, andT-cell receptors. For example, the antigen-binding domain may comprise:a single-chain variable fragment (scFv) derived from a monoclonalantibody; the binding domain from a natural receptor for the targetantigen; a peptide with sufficient affinity for the target ligand; asingle domain binder such as a camelid; an artificial binder single as aDarpin; or a single-chain derived from a T-cell receptor.

The term “ligand” is used synonymously with “antigen” to mean an entitywhich is specifically recognised and bound by the antigen-binding domainof the CCR.

Where the ligand is a tumour secreted factor, the antigen binding domainmay comprise an immunoglobulin-based antigen binding site, such as anscFv or a single domain binder.

Where the ligand is a chemokine, the antigen binding domain may comprisethe chemokine-binding portion of a natural receptor for the chemokine.

Ligand

The CCR of the present invention binds ligand.

The ligand may be a soluble ligand such as a tumour secreted factor or achemokine.

Alternatively, the ligand may be a membrane bound ligand, such as a cellsurface antigen.

The term “soluble ligand” is used to indicate a ligand or antigen whichis not part of or attached to a cell but which moves freely in theextracellular space, for example in a bodily fluid of the tissue ofinterest. The soluble ligand may exist in a cell-free state in theserum, plasma or other bodily fluid of an individual.

The soluble ligand may be associated with the presence or pathology of aparticular disease, such as cancer.

The soluble ligand may be part of the cancer secretome, i.e. thecollection of factors secreted by a tumour, be it from cancer stemcells, non-stem cells or the surrounding stroma. The soluble ligand maybe secreted or shed by tumour cells (see next section).

The soluble ligand may be characteristic of a disease or of diseasedtissue. It may be found exclusively, or at a higher level in a subjecthaving the disease vs a healthy subject; or in diseased tissue vshealthy tissue. The soluble ligand may be expressed at at least a2-fold, 5-fold, 10-fold, 100-fold, 1000-fold, 10,000-fold or 100,000fold higher level a subject having the disease vs a healthy subject; orin diseased tissue vs healthy tissue.

The terms “cell-surface antigen” and “cell-surface ligand” is usedsynonymously with “membrane-bound antigen” and “membrane-bound ligand”to mean a ligand which is attached to or expressed on the surface of thecell. The cell-surface ligand may, for example, be a transmembraneprotein.

The cell on which the cell-surface ligand is found may be a target cell,such as a cancer cell.

The cell-surface ligand may be associated with the presence or pathologyof a particular disease, such as cancer. Alternatively the cell-surfaceligand may be characteristic of the cell type of the target cell (e.g.B-cell) without being necessarily associated with the diseased state.

Where the cell-surface ligand is characteristic of a disease or ofdiseased tissue it may be found exclusively, or at a higher level on therelevant cells a subject having the disease vs a healthy subject; or indiseased tissue vs healthy tissue. The cell-surface ligand may beexpressed at at least a 2-fold, 5-fold, 10-fold, 100-fold, 1000-fold,10,000-fold or 100,000 fold higher level on a cell of a subject havingthe disease vs a healthy subject; or in diseased tissue vs healthytissue.

Tumour Secreted Factor

The ligand recognised by the CCR may be a soluble ligand secreted by orshedded from a tumour.

This “tumour secreted factor” may, for example, be prostate-specificantigen (PSA), carcinoembryonic antigen (CEA), vascular endothelialgrowth factor (VEGF) or Cancer Antigen -125 (CA-125).

The tumour secreted factor may be a soluble ligand which is not acytokine. The CCR of the present invention therefore grafts the bindingspecificity for a non-cytokine ligand on to the endodomain of a cytokinereceptor.

Prostate-Specific Antigen (PSA)

The soluble ligand may be prostate-specific antigen (PSA).

Prostate-specific antigen (PSA), also known as gamma-seminoprotein orkallikrein-3 (KLK3), is a glycoprotein enzyme encoded in humans by theKLK3 gene. PSA is a member of the kallikrein-related peptidase familyand is secreted by the epithelial cells of the prostate gland.

PSA is present in small quantities in the serum of men with healthyprostates, but is elevated in individuals with prostate cancer and otherprostate disorders.

PSA is a 237-residue glycoprotein and is activated by KLK2. Itsphysiological role is the liquefaction of the coagulum components of thesemen leading to liberation of spermatozoa. In cancer, PSA mayparticipate in the processes of neoplastic growth and metastasis.

PSA is a chymotrypsin-like serine protease with a typical His-Asp-Sertriad and a catalytic domain similar to those of otherkallikrein-related peptidases. The crystal structure of PSA has beenobtained i) in complex with the monoclonal antibody (mAb) 8G8F5 and ii)in a sandwich complex with two mAbs 5D5A5 and 5D3D11 (Stura et al (J.Mol. Biol. (2011) 414:530-544).

Various monoclonal antibodies are known, including clones 2G2-B2,2D8-E8, IgG1/K described in Bavat et al Avicenna J. Med. Biotechnol.2015, 7:2-7; and Leinonen (2004) 289:157-67.

The CCR of the present invention may, for example, comprise the 6 CDRsor the VH and/or VL domain(s) from a PSA-binding mAb such as 8G8F5,5D5A5 or 5D3D11.

Where the CCR comprises two antigen binding specificities, bindingdifferent epitopes on PSA, one may be bsed on, for example 5D3D11 andone may be based on, for example, 5D5A5.

The amino acid sequences for 5D3D11 and 5D5A5 VH and VL are given below.The complementarity determining regions (CDRs) are highlighted in bold.

5D3D11 VH (SEQ ID No. 11) QVQLQQSGPELVKPGASVKISCKVSGYAIS SSWMNWVKQRPGQGLEWIG RIYPGDGDTKYNGKFKD KATLTVDKSSSTAYMQLSSLTSVDSAVYFCARDGYRYYFDY WGQGTSVTVSS 5D3D11 VL (SEQ ID No. 12) DIVMTQTAPSVFVTPGESVSISCRSSKSLLHSNGNTYLY WFLQRPGQSP QLLIY RMSNLASGVPDRFSGSGSGTDFTLRISRVEAEDVGVYYC MQHLE YPVT FGAGTKVEIK 5D5A5 VH(SEQ ID No. 13) QVQLQQSGAELAKPGASVKMSCKTSGYSFS SYWMH WVKQRPGQGLEWIGYINPSTGYTENN Q KFKD KVTLTADKSSNTAYMQLNSLTSEDSAVYYCAR SGRLYFDVWGAGTTVTVSS 5D5A5 VL (SEQ ID No. 14) DIVLTQSPPSLAVSLGQRATISCRASESIDLYGFTFMH WYQQKPGQPP KILIY RASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYC QQTH EDPYT FGGGTKLEIKScFv based on 5D5A5 (SEQ ID No. 15)QVQLQQSGAELAKPGASVKMSCKTSGYSFSSYWMHVWKQRPGQGLEWIGYINPSTGYTENNQKFKDKVTLTADKSSNTAYMQLNSLTSEDSAVYYCARSGRLYFDVWGAGTTVIVSSGGGGSGGGGSGGGGSGGGGSDIVLTQSPPSLAVSLGQRATISCRASESIDLYGFTFMHWYQQKPGQPPKILIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQTHEDPYTFGGGTKL EIKScFv based on 5D3D11 (SEQ ID No. 16)QVQLQQSGPELVKPGASVKISCKVSGYAISSSWMNWVKQRPGQGLEWIGRIYPGDGDTKYNGKFKDKATLTVDKSSSTAYMQLSSLTSVDSAVYFCARDGYRYYFDYWGQGTSVTVSSGGGGSGGGGSGGGGSGGGGSDIVMTQTAPSVFVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTDFTLRISRVEAEDVGVYYCMQHLEYPVTFGAGT KVEIK

Where a cell comprises two CCRs, the antigen-binding domain of the firstCCR may comprise the 6 CDRs from 5D5A5 and the antigen-binding domain ofthe second CCR may comprise the 6 CDRs from 5D3D11.

The antigen-binding domain of the first CCR may comprise the VH and/orVL domain(s) from 5D5A5 or a variant thereof; and the antigen-bindingdomain of the second CCR may comprise the VH and/or VL domain(s) from5D3D11 or a variant thereof. Variant VH and VL domains may have at least80, 90, 95 or 99% identity to the sequences given above, provided thatthey retain PSA-binding activity.

A cell expressing a CCR which binds PSA may be useful in the treatmentof prostate cancer.

Carcinoembryonic Antigen (CEA) The soluble ligand may be CEA.

Carcinoembryonic antigen (CEA) describes a set of highly relatedglycoproteins involved in cell adhesion. CEA is normally produced ingastrointestinal tissue during fetal development, but the productionstops before birth. Therefore CEA is usually present only at very lowlevels in the blood of healthy adults. However, the serum levels areraised in some types of cancer, which means that it can be used as atumor marker in clinical tests.

CEA are glycosyl phosphatidyl inositol (GPI) cell surface anchoredglycoproteins whose specialized sialofucosylated glycoforms serve asfunctional colon carcinoma L-selectin and E-selectin ligands, which maybe critical to the metastatic dissemination of colon carcinoma cells.Immunologically they are characterized as members of the CD66 cluster ofdifferentiation.

CEA and related genes make up the CEA family belonging to theimmunoglobulin superfamily. In humans, the carcinoembryonic antigenfamily consists of 29 genes, 18 of which are normally expressed. Thefollowing is a list of human genes which encode carcinoembryonicantigen-related cell adhesion proteins: CEACAM1, CEACAM3, CEACAM4,CEACAM5, CEACAM6, CEACAM7, CEACAM8, CEACAM16, CEACAM18, CEACAM19,CEACAM20, CEACAM21

Various antibodies which target CEA are described in WO 2011/034660.

A cell expressing a CCR against CEA may be useful in the treatment of,for example, colorectal cancer.

Vascular Endothelial Growth Factor (VEGF)

The soluble ligand may be VEGF.

Vascular endothelial growth factor (VEGF) is a signal protein producedby cells that stimulates vasculogenesis and angiogenesis. It is part ofthe system that restores the oxygen supply to tissues when bloodcirculation is inadequate. Serum concentration of VEGF is high inbronchial asthma and diabetes mellitus. VEGF's normal function is tocreate new blood vessels during embryonic development, new blood vesselsafter injury, muscle following exercise, and new vessels (collateralcirculation) to bypass blocked vessels.

When VEGF is overexpressed, it can contribute to disease. Solid cancerscannot grow beyond a limited size without an adequate blood supply;cancers that can express VEGF are able to grow and metastasize.

VEGF is a sub-family of the platelet-derived growth factor family ofcystine-knot growth factors. They are important signaling proteinsinvolved in both vasculogenesis (the de novo formation of the embryoniccirculatory system) and angiogenesis (the growth of blood vessels frompre-existing vasculature).

The VEGF family comprises in mammals five members: VEGF-A, placentagrowth factor (PGF), VEGF-B, VEGF-C and VEGF-D.

Various antibodies to VEGF are known, such as bevacizumab (Avastin) andRanibizumab (Lucentis).

Cancer Antigen 125 (CA-125)

CA-125 is associated with ovarian cancer and is the most frequently usedbiomarker for ovarian cancer detection. While CA-125 is best known as amarker for ovarian cancer, it may also be elevated in other cancers,including endometrial cancer, fallopian tube cancer, lung cancer, breastcancer and gastrointestinal cancer.

The sequence of human CA-125 (also known as mucin-16) is available fromNCBI, Accession No. 078966.

A number of CA125-binding monoclonal antibodies are known, includingOC125 and M11 (Nustad et al 1996, Tumour Biol. 17:196-329). In thisstudy the specificity of 26 monoclonal antibodies against the CA 125antigen was investigated. It was found that the CA 125 antigen carriesonly two major antigenic domains, which classifies the antibodies asOC125-like (group A) or M11-like (group B).

The chimeric cytokine receptor of the present invention may comprise anantigen-binding domain from such an antibody. A cell comprising such aCCR may be useful in the treatment of, for example, ovarian cancer.

The tumour secreted factor (or, if in a membrane-bound form, thetransmembrane protein) may be selected from the following non-exhaustivelist:

ALK gene rearrangements and overexpression giving mutated forms of ALKproteins

Alpha-fetoprotein (AFP)

Beta-2-microglobulin (B2M)Beta-human chorionic gonadotropin (Beta-hCG)BRAF V600 mutations giving mutated B-REF protein

C-kit/CD117 CA15-3/CA27.29 CA19-9 Calcitonin CD20 Chromogranin A (CgA)

Cytokeratin fragment 21-1EGFR gene mutation analysisEstrogen receptor (ER)/progesterone receptor (PR)

Fibrin/fibrinogen HE4

HER2/neu gene amplification or protein overexpression

Immunoglobulins

KRAS gene mutation analysisLactate dehydrogenaseNeuron-specific enolase (NSE)Nuclear matrix protein 22Programmed death ligand 1 (PD-L1)

Thyroglobulin

Urokinase plasminogen activator (uPA) and plasminogen activatorinhibitor (PAI-1)

Chemokine

Chemokines are chemotactic cytokines. Cell migration is guided bychemokine gradients embedded and immobilized in extracellular matrix.The positively charged chemokines like CXCL12 bind to negatively chargedECM molecules. These gradients provide tracks for cancer cell and immunecell homing. The action on T cells seems to be inhibitory for the homingof cytotoxic T cells, while regulatory T cells appear to be attracted.

Chemokines are approximately 8-10 kilodaltons in mass and have fourcysteine residues in conserved locations which are key to forming their3-dimensional shape.

Some chemokines are considered pro-inflammatory and can be inducedduring an immune response to recruit cells of the immune system to asite of infection, while others are considered homeostatic and areinvolved in controlling the migration of cells during normal processesof tissue maintenance or development.

Chemokines have been classified into four main subfamilies: CXC, CC,CX3C and XC. All of these proteins exert their biological effects byinteracting with G protein-linked transmembrane receptors calledchemokine receptors that are selectively found on the surfaces of theirtarget cells.

The major role of chemokines is to act as a chemoattractant to guide themigration of cells. Cells that are attracted by chemokines follow asignal of increasing chemokine concentration towards the source of thechemokine. Some chemokines control cells of the immune system duringprocesses of immune surveillance, such as directing lymphocytes to thelymph nodes so they can screen for invasion of pathogens by interactingwith antigen-presenting cells residing in these tissues. Otherchemokines are inflammatory and are released from a wide variety ofcells in response to bacterial infection, viruses and other agents.Their release is often stimulated by pro-inflammatory cytokines such asinterleukin 1. Inflammatory chemokines function mainly aschemoattractants for leukocytes, recruiting monocytes, neutrophils andother effector cells from the blood to sites of infection or tissuedamage. Certain inflammatory chemokines activate cells to initiate animmune response or promote wound healing. They are released by manydifferent cell types and serve to guide cells of both innate immunesystem and adaptive immune system.

CC Chemokines

The CC chemokine (or β-chemokine) proteins have two adjacent cysteines(amino acids), near their amino terminus. There have been at least 27distinct members of this subgroup reported for mammals, called CCchemokine ligands (CCL)-1 to -28; CCL10 is the same as CCL9. Chemokinesof this subfamily usually contain four cysteines (C4-CC chemokines), buta small number of CC chemokines possess six cysteines (C6-CCchemokines). C6-CC chemokines include CCL1, CCL15, CCL21, CCL23 andCCL28. CC chemokines induce the migration of monocytes and other celltypes such as NK cells and dendritic cells.

Examples of CC chemokine include monocyte chemoattractant protein-1(MCP-1 or CCL2) which induces monocytes to leave the bloodstream andenter the surrounding tissue to become tissue macrophages.

CCL5 (or RANTES) attracts cells such as T cells, eosinophils andbasophils that express the receptor CCR5.

CXC Chemokines

The two N-terminal cysteines of CXC chemokines (or α-chemokines) areseparated by one amino acid, represented in this name with an “X”. Therehave been 17 different CXC chemokines described in mammals, that aresubdivided into two categories, those with a specific amino acidsequence (or motif) of glutamic acid-leucine-arginine (or ELR for short)immediately before the first cysteine of the CXC motif (ELR-positive),and those without an ELR motif (ELR-negative). ELR-positive CXCchemokines specifically induce the migration of neutrophils, andinteract with chemokine receptors CXCR1 and CXCR2.

C Chemokines

The third group of chemokines is known as the C chemokines (or γchemokines), and is unlike all other chemokines in that it has only twocysteines; one N-terminal cysteine and one cysteine downstream. Twochemokines have been described for this subgroup and are called XCL1(lymphotactin-α) and XCL2 (Iymphotactin-β).

CX3C Chemokine

CX3C chemokines have three amino acids between the two cysteines. Theonly CX3C chemokine discovered to date is called fractalkine (orCX3CL1). It is both secreted and tethered to the surface of the cellthat expresses it, thereby serving as both a chemoattractant and as anadhesion molecule.

Chemokine receptors are G protein-coupled receptors containing 7transmembrane domains that are found on the surface of leukocytes.Approximately 19 different chemokine receptors have been characterizedto date, which are divided into four families depending on the type ofchemokine they bind; CXCR that bind CXC chemokines, CCR that bind CCchemokines, CX3CR1 that binds the sole CX3C chemokine (CX3CL1), and XCR1that binds the two XC chemokines (XCL1 and XCL2). They share manystructural features; they are similar in size (with about 350 aminoacids), have a short, acidic N-terminal end, seven helical transmembranedomains with three intracellular and three extracellular hydrophilicloops, and an intracellular C-terminus containing serine and threonineresidues important for receptor regulation. The first two extracellularloops of chemokine receptors each has a conserved cysteine residue thatallow formation of a disulfide bridge between these loops. G proteinsare coupled to the C-terminal end of the chemokine receptor to allowintracellular signaling after receptor activation, while the N-terminaldomain of the chemokine receptor determines ligand binding specificity.

CXCL12

CXCL12 is strongly chemotactic for lymphocytes. CXCL12 plays animportant role in angiogenesis by recruiting endothelial progenitorcells (EPCs) from the bone marrow through a CXCR4 dependent mechanism.It is this function of CXCL12 that makes it a very important factor incarcinogenesis and the neovascularisation linked to tumour progression.CXCL12 also has a role in tumour metastasis where cancer cells thatexpress the receptor CXCR4 are attracted to metastasis target tissuesthat release the ligand, CXCL12.

The receptor for CXCL12 is CXCR4. The CCR of the present invention maycomprise the CXCL12-binding domain from CXCR4 linked to an endodomainderived from a cytokine receptor, such as the IL-2 receptor.

CXCR4 coupled expression of IL2 would support engraftment of therapeuticT cell for cancer therapies. In multiple myeloma, a cell expressing sucha CCR may mobilize cells and change the bone marrow environment. Suchcells also have uses in the treatment of solid cancers by modifying thesolid tumour microenvironment.

The amino acid sequence for CXCR4 is shown below as SEQ ID No. 17

SEQ ID No. 17   1msiplpllqi ytsdnyteem gsgdydsmke pcfreenanf nkiflptiys iifltgivgn  61glvilvmgyq kklrsmtdky rlhlsvadll fvitlpfwav davanwyfgn flckavhviy 121tvnlyssvli lafisldryl aivhatnsqr prkllaekvv yvgvwipall Itipdfifan 181vseaddryic drfypndlwv vvfqfqhimv glilpgivil scyciiiskl shskghqkrk 241alkttvilil affacwlpyy igisidsfil leiikqgcef entvhkwisi tealaffhcc 301lnpilyafIg akfktsaqha ltsysrgssl kilskgkrgg hssysteses ssfhss

CXCR7 also binds CXCL12.

CCL2

The chemokine (C—C motif) ligand 2 (CCL2) is also referred to asmonocyte chemotactic protein 1 (MCP1) and small inducible cytokine A2.CCL2 recruits monocytes, memory T cells, and dendritic cells to thesites of inflammation produced by either tissue injury or infection.

CCR2 and CCR4 are two cell surface receptors that bind CCL2.

CCR2 has the amino acid sequence shown as SEQ ID No. 18

SEQ ID No. 18   1mlstsrsrfi rntnesgeev ttffdydyga pchkfdvkqi gaqllpplys lvfifgfvgn  61mlvvlilinc kklkcltdiy llnlaisdll flitlplwah saanewvfgn amcklftgly 121higyfggiff iilltidryl aivhavfalk artvtfgvvt svitwlvavf asvpgiiftk 181cqkedsvyvc gpyfprgwnn fhtimrnilg lvIpllimvi cysgilktll rcrnekkrhr 241avrviftimi vyflfwtpyn ivillntfqe ffglsncest sqldqatqvt etlgmthcci 301npiiyafvge kfrslfhial gcriaplqkp vcggpgvrpg knvkvttqgl ldgrgkgksi 361grapeaslqd kega

CCR4 has the amino acid sequence shown as SEQ ID No. 19.

SEQ ID No. 19   1mnptdiadtt ldesiysnyy lyesipkpct kegikafgel flpplyslvf vfgllgnsvv  61vlvlfkykrl rsmtdvylln laisdllfvf slpfwgyyaa dqwvfglglc kmiswmylvg 121fysgiffvml msidrylaiv havfslrart ltygvitsla twsvavfasl pgflfstcyt 181ernhtycktk yslnsttwkv lssleinilg lviplgimlf cysmiirtlq hcknekknka 241vkmifavvvl flgfwtpyni vlfletivel evlqdctfer yldyaiqate tlafvhccln 301piiyfflgek frkyilqlfk tcrglfylcq ycgllqiysa dtpsssytqs tmdhdlhdal

The CCR of the present invention may comprise the CCL2 binding site ofCCR2 or CCR4 in its ligand binding domain.

Cell-Surface Antigen

The ligand may be a cell-surface antigen, such as a transmembraneprotein.

The cell surface antigen may be CD22.

CD22, or cluster of differentiation-22, is a molecule belonging to theSIGLEC family of lectins. It is found on the surface of mature B cellsand to a lesser extent on some immature B cells. Generally speaking,CD22 is a regulatory molecule that prevents the overactivation of theimmune system and the development of autoimmune diseases.

CD22 is a sugar binding transmembrane protein, which specifically bindssialic acid with an immunoglobulin (Ig) domain located at itsN-terminus. The presence of Ig domains makes CD22 a member of theimmunoglobulin superfamily. CD22 functions as an inhibitory receptor forB cell receptor (BCR) signalling.

Increased expression of CD22 is seen in non-Hodgkin and other lymphomas.Various monoclonal antibodies targeting CD22 are known, includingepratuzumab, inotuzumab ozogamicin, m971 and m972.

Chimeric Antigen Receptors (CAR)

The cell of the present invention may also comprise one or more chimericantigen receptor(s). The CAR(s) may be specific for a tumour-associatedantigen.

Classical CARs are chimeric type I trans-membrane proteins which connectan extracellular antigen-recognizing domain (binder) to an intracellularsignalling domain (endodomain). The binder is typically a single-chainvariable fragment (scFv) derived from a monoclonal antibody (mAb), butit can be based on other formats which comprise an antibody-like orligand-based antigen binding site. A trans-membrane domain anchors theprotein in the cell membrane and connects the spacer to the endodomain.

Early CAR designs had endodomains derived from the intracellular partsof either the γ chain of the FcεR1 or CD3. Consequently, these firstgeneration receptors transmitted immunological signal 1, which wassufficient to trigger T-cell killing of cognate target cells but failedto fully activate the T-cell to proliferate and survive. To overcomethis limitation, compound endodomains have been constructed: fusion ofthe intracellular part of a T-cell co-stimulatory molecule to that ofCD3ζ results in second generation receptors which can transmit anactivating and co-stimulatory signal simultaneously after antigenrecognition. The co-stimulatory domain most commonly used is that ofCD28. This supplies the most potent co-stimulatory signal-namelyimmunological signal 2, which triggers T-cell proliferation. Somereceptors have also been described which include TNF receptor familyendodomains, such as the closely related OX40 and 41BB which transmitsurvival signals. Even more potent third generation CARs have now beendescribed which have endodomains capable of transmitting activation,proliferation and survival signals.

CAR-encoding nucleic acids may be transferred to T cells using, forexample, retroviral vectors. In this way, a large number ofantigen-specific T cells can be generated for adoptive cell transfer.When the CAR binds the target-antigen, this results in the transmissionof an activating signal to the T-cell it is expressed on. Thus the CARdirects the specificity and cytotoxicity of the T cell towards cellsexpressing the targeted antigen.

The cell of the present invention may comprise one or more CAR(s).

The CAR(s) may comprise an antigen-binding domain, a spacer domain, atransmembrane domain and an endodomain. The endodomain may comprise orassociate with a domain which transmit T-cell activation signals.

Car Antigen Binding Domain

The antigen-binding domain is the portion of a CAR which recognizesantigen.

Numerous antigen-binding domains are known in the art, including thosebased on the antigen binding site of an antibody, antibody mimetics, andT-cell receptors. For example, the antigen-binding domain may comprise:a single-chain variable fragment (scFv) derived from a monoclonalantibody; a natural ligand of the target antigen; a peptide withsufficient affinity for the target; a single domain binder such as acamelid; an artificial binder single as a Darpin; or a single-chainderived from a T-cell receptor.

The term “ligand” is used synonymously with “antigen” to mean an entitywhich is specifically recognised and bound by the antigen-binding domainof a CAR.

Cell Surface Antigen

The CAR may recognise a cell-surface antigen, i.e. an entity, such as atransmembrane protein which is expressed on the surface of a targetcell, such as a tumour cell.

The CAR may specifically bind a tumour-associated cell-surface antigen.

Various tumour associated antigens (TAA) are known, some of which areshown in Table 1. The antigen-binding domain used in the presentinvention may be a domain which is capable of binding a TAA as indicatedtherein.

TABLE 1 Cancer type TAA Diffuse Large B-cell Lymphoma CD19, CD20, CD22Breast cancer ErbB2, MUC1 AML CD13, CD33 Neuroblastoma GD2, NCAM, ALK,GD2 B-CLL CD19, CD52, CD160 Colorectal cancer Folate binding protein,CA-125 Chronic Lymphocytic Leukaemia CD5, CD19 Glioma EGFR, VimentinMultiple myeloma BCMA, CD138 Renal Cell Carcinoma Carbonic anhydrase IX,G250 Prostate cancer PSMA Bowel cancer A33

Where the CAR recognises a B-cell lymphoma or leukemia antigen (such asCD19, CD20, CD52, CD160 or CD5), the CCR may recognise another B-cellantigen, such as CD22.

Prostate-Cancer Associated Antigens

The CAR may specifically bind a cell-surface antigen associated withprostate cancer, such as prostate stem cell antigen (PSCA) orprostate-specific membrane antigen (PSMA).

PSCA is a glycosylphosphatidylinositol-anchored cell membraneglycoprotein. It is is up-regulated in a large proportion of prostatecancers and is also detected in cancers of the bladder and pancreas.

Various anti-PSCA antibodies are known, such as 7F5 (Morgenroth et al(Prostate (2007) 67:1121-1131); 1G8 (Hillerdal et al (2014) BMC Cancer14:30); and Ha1-4.117 (Abate-Daga et al (2014) 25:1003-1012).

The CCR-expressing cell of the invention may also express an anti-PSCACAR which may comprise an antigen binding domain based on one of theseantibodies.

PSMA is is a zinc metalloenzyme that resides in membranes. PSMA isstrongly expressed in the human prostate, being a hundredfold greaterthan the expression in most other tissues. In cancer, it is upregulatedin expression and has been called the second-most-upregulated gene inprostate cancer, with increase of 8- to 12-fold over the noncancerousprostate. In addition to the expression in the human prostate andprostate cancer, PSMA is also found to be highly expressed in tumorneovasculature but not normal vasculature of all types of solid tumors,such as kidney, breast, colon, etc.

Various anti-PSMA antibodies are known, such as 7E11, J591, J415, andHybritech PEQ226.5 and PM2J004.5 each of which binds a distinct epitopeof PSMA (Chang et al (1999) Cancer Res 15:3192-8).

The CCR-expressing cell of the invention may also express an anti-PSMACAR which may comprise an antigen binding domain based on one of theseantibodies.

For example, the CCR may comprise an scFv based on J591, having thesequence shown as SEQ ID No. 20.

(J591 scFv) SEQ ID No. 20EVQLQQSGPELKKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAAGWNFDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCQQYNSYPLTFGAGTMLDLKR

Car Transmembrane Domain

The transmembrane domain is the sequence of a CAR that spans themembrane. It may comprise a hydrophobic alpha helix. The CARtransmembrane domain may be derived from CD28, which gives good receptorstability.

Car Signal Peptide

The CAR and CCR described herein may comprise a signal peptide so thatwhen it/they is expressed in a cell, such as a T-cell, the nascentprotein is directed to the endoplasmic reticulum and subsequently to thecell surface, where it is expressed.

The core of the signal peptide may contain a long stretch of hydrophobicamino acids that has a tendency to form a single alpha-helix. The signalpeptide may begin with a short positively charged stretch of aminoacids, which helps to enforce proper topology of the polypeptide duringtranslocation. At the end of the signal peptide there is typically astretch of amino acids that is recognized and cleaved by signalpeptidase. Signal peptidase may cleave either during or after completionof translocation to generate a free signal peptide and a mature protein.The free signal peptides are then digested by specific proteases.

The signal peptide may be at the amino terminus of the molecule.

The signal peptide may comprise the sequence shown as SEQ ID No. 21, 22or 23 or a variant thereof having 5, 4, 3, 2 or 1 amino acid mutations(insertions, substitutions or additions) provided that the signalpeptide still functions to cause cell surface expression of the CAR.

SEQ ID No. 21: MGTSLLCWMALCLLGADHADG

The signal peptide of SEQ ID No. 21 is compact and highly efficient andis derived from TCR beta chain. It is predicted to give about 95%cleavage after the terminal glycine, giving efficient removal by signalpeptidase.

SEQ ID No. 22: MSLPVTALLLPLALLLHAARP

The signal peptide of SEQ ID No. 22 is derived from IgG1.

SEQ ID No. 23: MAVPTQVLGLLLLWLTDARC

The signal peptide of SEQ ID No. 23 is derived from CD8a.

Car Endodomain

The endodomain is the portion of a classical CAR which is located on theintracellular side of the membrane.

The endodomain is the signal-transmission portion of a classical CAR.After antigen recognition by the antigen binding domain, individual CARmolecules cluster, native CD45 and CD148 are excluded from the synapseand a signal is transmitted to the cell.

The CAR endodomain may be or comprise an intracellular signallingdomain. In an alternative embodiment, the endodomain of the present CARmay be capable of interacting with an intracellular signalling moleculewhich is present in the cytoplasm, leading to signalling.

The intracellular signalling domain or separate intracellular signallingmolecule may be or comprise a T cell signalling domain.

The most commonly used signalling domain component is that of CD3-zetaendodomain, which contains 3 ITAMs. This transmits an activation signalto the T cell after antigen is bound. CD3-zeta may not provide a fullycompetent activation signal and additional co-stimulatory signalling maybe needed. For example, chimeric CD28 and OX40 can be used with CD3-Zetato transmit a proliferative/survival signal, or all three can be usedtogether.

The CAR may comprise the CD3-Zeta endodomain alone, the CD3-Zetaendodomain with that of either CD28 or OX40 or the CD28 endodomain andOX40 and CD3-Zeta endodomain.

The CAR endodomain may comprise one or more of the following: an ICOSendodomain, a CD27 endodomain, a BTLA endodomain, a CD30 endodomain, aGITR endodomain and an HVEM endodomain.

The endomain may comprise the sequence shown as SEQ ID No. 24 to 32 or avariant thereof having at least 80% sequence identity.

CD3 Z endodomain SEQ ID No. 24RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPRCD28 and CD3 Zeta endodomains SEQ ID No. 25SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPRCD28, OX40 and CD3 Zeta endodomains SEQ ID No. 26SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR ICOS endodomain SEQ ID No. 27CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL CD27 endodomain SEQ ID No. 28QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP BTLA endodomainSEQ ID No. 29 RRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDNDPDLCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVK EAPTEYASICVRSCD30 endodomain SEQ ID No. 30HRRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTEPVAEERGLMSQPLMETCHSVGAAYLESLPLQDASPAGGPSSPRDLPEPRVSTEHTNNKIEKIYIMKADTVIVGTVKAELPEGRGLAGPAEPELEEELEADHTPHYPEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK GITR endodomain SEQ ID No. 31QLGLHIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEE KGRLGDLWVHVEM endodomain SEQ ID No. 32CVKRRKPRGDWKVIVSVQRKRQEAEGEATVIEALQAPPDVTTVAVEET IPSFTGRSPNH

A variant sequence may have at least 80%, 85%, 90%, 95%, 98% or 99%sequence identity to SEQ ID No. 24 to 32, provided that the sequenceprovides an effective intracellular signalling domain.

Nucleic Acid

The present invention also provides a nucleic acid encoding a CCR of theinvention.

The nucleic acid may have the structure:

AgB-spacer-TM-endo

in which

AgB1 is a nucleic acid sequence encoding the antigen-binding domain ofthe CCR; spacer 1 is a nucleic acid sequence encoding the spacer of theCCR;

TM1 is a a nucleic acid sequence encoding the transmembrane domain ofthe CCR; endo 1 is a nucleic acid sequence encoding the endodomain ofthe CCR.

Nucleic Acid Construct

The present invention further provides a nucleic acid construct whichcomprises a first nucleic acid sequence encoding a first CCR as definedin connection with the first aspect of the invention; and a secondnucleic acid sequence encoding a second CCR as defined in connectionwith the first aspect of the invention.

The nucleic acid construct may have the following structure:

AgB1-spacer1-TM1-endo1-coexpr-AgB2-spacer2-TM2-endo2

in which

AgB1 is a nucleic acid sequence encoding the antigen-binding domain ofthe first CCR;

spacer 1 is a nucleic acid sequence encoding the spacer of the firstCCR;

TM1 is a a nucleic acid sequence encoding the transmembrane domain ofthe first CCR;

endo 1 is a nucleic acid sequence encoding the endodomain of the firstCCR;

coexpr is a nucleic acid sequence enabling co-expression of both CCRs

AgB2 is a nucleic acid sequence encoding the antigen-binding domain ofthe second CCR;

spacer 2 is a nucleic acid sequence encoding the spacer of the secondCCR;

TM2 is a a nucleic acid sequence encoding the transmembrane domain ofthe second CCR;

endo 2 is a nucleic acid sequence encoding the endodomain of the secondCCR.

When the nucleic acid construct is expressed in a cell, such as aT-cell, it encodes a polypeptide which is cleaved at the cleavage sitesuch that the first and second CCRs are co-expressed at the cellsurface.

The first and second CCRs may bind distinct epitopes on the sameantigen.

The first and second CCRs may have complementary endodomains e.g. onederived from the α or β chain of a cytokine receptor and one derivedfrom the γ chain of the same cytokine receptor.

The present invention also provides a nucleic acid construct encoding aCCR of the invention and a CAR. Such a construct may have the structure:

CCRAgB-CCRspacer-CCRTM-CCRendo-coexpr-CARAgB-CARspacer-CARTM-CARendo

or

CARAgB-CARspacer-CARTM-CARendo-coexpr-CCRAgB-CCRspacer-CCRTM-CCRendo

in which

CCRAgB is a nucleic acid sequence encoding the antigen-binding domain ofthe CCR;

CCRspacer is a nucleic acid sequence encoding the spacer of the CCR;

CCRTM is a a nucleic acid sequence encoding the transmembrane domain ofthe CCR;

CCRendo is a nucleic acid sequence encoding the endodomain of the CCR;

coexpr is a nucleic acid sequence enabling co-expression of both the CCRand the CAR

CARAgB is a nucleic acid sequence encoding the antigen-binding domain ofthe CAR;

CARspacer is a nucleic acid sequence encoding the spacer of the CAR;

CARTM is a nucleic acid sequence encoding the transmembrane domain ofthe CAR; and

CARendo is a nucleic acid sequence encoding the endodomain of the CAR.

The present invention also provides a nucleic acid construct encoding afirst and a second CCR of the invention and a CAR. The first and secondCCRs may bind separate epitopes on the same antigen. Such a constructmay have the structure:

(i)CCRAgB1-CCRspacer1-CCRTM1-CCRendo1-coexpr1-CCRAgB2-CCRspacer2-CCRTM2-CCRendo2-coexpr2-CARAgB-CARspacer-CARTM-CARendo,

(ii)CCRAgB1-CCRspacer1-CCRTM1-CCRendo1-coexpr1-CARAgB-CARspacer-CARTM-CARendo-coexpr2-CCRAgB2-CCRspacer2-CCRTM2-CCRendo2;or

(iii)CARAgB-CARspacer-CARTM-CARendo-coexpr1-CCRAgB1-CCRspacer1-CCRTM1-CCRendo1-coexpr2-CCRAgB2-CCRspacer2-CCRTM2-CCRendo2;

in which

CCRAgB1 is a nucleic acid sequence encoding the antigen-binding domainof the first CCR;

CCRspacer1 is a nucleic acid sequence encoding the spacer of the firstCCR;

CCRTM1 is a nucleic acid sequence encoding the transmembrane domain ofthe first CCR;

CCRendo1 is a nucleic acid sequence encoding the endodomain of the firstCCR;

CCRAgB2 is a nucleic acid sequence encoding the antigen-binding domainof the second CCR;

CCRspacer2 is a nucleic acid sequence encoding the spacer of the secondCCR;

CCRTM2 is a nucleic acid sequence encoding the transmembrane domain ofthe second CCR;

CCRendo2 is a nucleic acid sequence encoding the endodomain of thesecond CCR;

Coexpr1 and coexpr2 are nucleic acid sequences enabling co-expression ofthe two flanking sequences;

CARAgB is a nucleic acid sequence encoding the antigen-binding domain ofthe CAR;

CARspacer is a nucleic acid sequence encoding the spacer of the CAR;

CARTM is a nucleic acid sequence encoding the transmembrane domain ofthe CAR; and

CARendo is a nucleic acid sequence encoding the endodomain of the CAR.

As used herein, the terms “polynucleotide”, “nucleotide”, and “nucleicacid” are intended to be synonymous with each other.

It will be understood by a skilled person that numerous differentpolynucleotides and nucleic acids can encode the same polypeptide as aresult of the degeneracy of the genetic code. In addition, it is to beunderstood that skilled persons may, using routine techniques, makenucleotide substitutions that do not affect the polypeptide sequenceencoded by the polynucleotides described here to reflect the codon usageof any particular host organism in which the polypeptides are to beexpressed.

Nucleic acids according to the invention may comprise DNA or RNA. Theymay be single-stranded or double-stranded. They may also bepolynucleotides which include within them synthetic or modifiednucleotides. A number of different types of modification tooligonucleotides are known in the art. These include methylphosphonateand phosphorothioate backbones, addition of acridine or polylysinechains at the 3′ and/or 5′ ends of the molecule. For the purposes of theuse as described herein, it is to be understood that the polynucleotidesmay be modified by any method available in the art. Such modificationsmay be carried out in order to enhance the in vivo activity or life spanof polynucleotides of interest.

The terms “variant”, “homologue” or “derivative” in relation to anucleotide sequence include any substitution of, variation of,modification of, replacement of, deletion of or addition of one (ormore) nucleic acid from or to the sequence.

In the structure above, “coexpr” is a nucleic acid sequence enablingco-expression of both first and second CARs. It may be a sequenceencoding a cleavage site, such that the nucleic acid construct producescomprises two or more CCRs, or a CCR and a CAR, joined by a cleavagesite(s). The cleavage site may be self-cleaving, such that when thepolypeptide is produced, it is immediately cleaved into individualpeptides without the need for any external cleavage activity.

The cleavage site may be any sequence which enables the first and secondCCRs, or CCR and CAR, to become separated.

The term “cleavage” is used herein for convenience, but the cleavagesite may cause the peptides to separate into individual entities by amechanism other than classical cleavage. For example, for theFoot-and-Mouth disease virus (FMDV) 2A self-cleaving peptide (seebelow), various models have been proposed for to account for the“cleavage” activity: proteolysis by a host-cell proteinase,autoproteolysis or a translational effect (Donnelly et al (2001) J. Gen.Virol. 82:1027-1041). The exact mechanism of such “cleavage” is notimportant for the purposes of the present invention, as long as thecleavage site, when positioned between nucleic acid sequences whichencode proteins, causes the proteins to be expressed as separateentities.

The cleavage site may be a furin cleavage site.

Furin is an enzyme which belongs to the subtilisin-like proproteinconvertase family. The members of this family are proprotein convertasesthat process latent precursor proteins into their biologically activeproducts. Furin is a calcium-dependent serine endoprotease that canefficiently cleave precursor proteins at their paired basic amino acidprocessing sites. Examples of furin substrates include proparathyroidhormone, transforming growth factor beta 1 precursor, proalbumin,pro-beta-secretase, membrane type-1 matrix metalloproteinase, betasubunit of pro-nerve growth factor and von Willebrand factor. Furincleaves proteins just downstream of a basic amino acid target sequence(canonically, Arg-X-(Arg/Lys)-Arg′) and is enriched in the Golgiapparatus.

The cleavage site may be a Tobacco Etch Virus (TEV) cleavage site.

TEV protease is a highly sequence-specific cysteine protease which ischymotrypsin-like proteases. It is very specific for its target cleavagesite and is therefore frequently used for the controlled cleavage offusion proteins both in vitro and in vivo. The consensus TEV cleavagesite is ENLYFQ\S (where ‘\’ denotes the cleaved peptide bond). Mammaliancells, such as human cells, do not express TEV protease. Thus inembodiments in which the present nucleic acid construct comprises a TEVcleavage site and is expressed in a mammalian cell—exogenous TEVprotease must also expressed in the mammalian cell.

The cleavage site may encode a self-cleaving peptide.

A ‘self-cleaving peptide’ refers to a peptide which functions such thatwhen the polypeptide comprising the proteins and the self-cleavingpeptide is produced, it is immediately “cleaved” or separated intodistinct and discrete first and second polypeptides without the need forany external cleavage activity.

The self-cleaving peptide may be a 2A self-cleaving peptide from anaphtho- or a cardiovirus. The primary 2A/2B cleavage of the aptho- andcardioviruses is mediated by 2A “cleaving” at its own C-terminus. Inapthoviruses, such as foot-and-mouth disease viruses (FM DV) and equinerhinitis A virus, the 2A region is a short section of about 18 aminoacids, which, together with the N-terminal residue of protein 2B (aconserved proline residue) represents an autonomous element capable ofmediating “cleavage” at its own C-terminus (Donelly et al (2001) asabove).

“2A-like” sequences have been found in picornaviruses other than aptho-or cardioviruses, ‘picornavirus-like’ insect viruses, type C rotavirusesand repeated sequences within Trypanosoma spp and a bacterial sequence(Donnelly et al (2001) as above). The cleavage site may comprise one ofthese 2A-like sequences, such as:

(SEQ ID No. 33) YHADYYKQRLIHDVEMNPGP (SEQ ID No. 34)HYAGYFADLLIHDIETNPGP (SEQ ID No. 35) QCTNYALLKLAGDVESNPGP(SEQ ID No. 36) ATNFSLLKQAGDVEENPGP (SEQ ID No. 37) AARQMLLLLSGDVETNPGP(SEQ ID No. 38) RAEGRGSLLTCGDVEENPGP (SEQ ID No. 39)TRAEIEDELIRAGIESNPGP (SEQ ID No. 40) TRAEIEDELIRADIESNPGP(SEQ ID No. 41) AKFQIDKILISGDVELNPGP (SEQ ID No. 42)SSIIRTKMLVSGDVEENPGP (SEQ ID No. 43) CDAQRQKLLLSGDIEQNPGP(SEQ ID No. 44) YPIDFGGFLVKADSEFNPGP

The cleavage site may comprise the 2A-like sequence shown as SEQ ID No.38 (RAEGRGSLLTCGDVEENPGP).

The present invention also provides a kit comprising one or more nucleicacid sequence(s) encoding first and second CCRs according to the firstaspect of the present invention, or one or more CCR(s) according to theinvention and one or more CAR(s).

SEQ ID NOS 45 and 46 give the complete amino acid sequences of a fusionbetween and anti-PSMA CAR and an anti-PSA CCR. Subheadings are given tolabel each portion of the sequence but in practice the various elementsare connected giving one continuous sequence.

The nucleic acid construct of the invention may encode a fusion proteinas shown in SEQ ID No. 45 or 46.

SEQ ID NO. 45—Illustrative construct with IL-2R beta chain

Signal sequence derived from human CD8a: MSLPVTALLLPLALLLHAAscFv aPSMA (J591 H/L) EVQLQQSGPELKKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAAGWNFDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKWYWASTRHTGVPDRFTGSGSGTDFILTITNVQSEDLADYFCQQYNSYPLTFGAGTMLDLKR Linker SDPAHuman IgG1 Fc spacer (HCH2CH3pvaa):EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Transmembrane derived from human CD28:FWVLVVVGGVLACYSLLVTVAFIIFWV Endodomain derived from TCRz:RRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKD TYDALHMQALPPR2A peptide from Thosea asigna virus capsid protein: RAEGRGSLLTCGDVEENPGPSignal sequence derived from mouse kappa VIII: METDTLILWVLLLLVPGSTGscFv aPSA (5D5A5 H/L): QVQLQQSGAELAKPGASVKMSCKTSGYSFSSYWMHWVKQRPGQGLEWIGYINPSTGYTENNQKFKDKVTLTADKSSNTAYMQLNSLTSEDSAVYYCARSGRLYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIVLTQSPPSLAVSLGQRATISCRASESIDLYGFTFMHWYQQKPGQPPKILIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQTHEDPYTFGGGTKL EIK Linker: SDPAHuman CD8aSTK spacer: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDITransmembrane derived from human common gamma  chain:VVISVGSMGLIISLLCVYFWL Endodomain derived from human common gamma  chain:ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET2A peptide from equine rhinitis A virus  polyprotein:QCTNYALLKLAGDVESNPGP Signal sequence derived from mouse kappa VIII:METDTLILWVLLLLVPGSTG scFv aPSA (5D3D11 H/L):QVQLQQSGPELVKPGASVKISCKVSGYAISSSWMNWVKQRPGQGLEWIGRIYPGDGDTKYNGKFKDKATLTVDKSSSTAYMQLSSLTSVDSAVYFCARDGYRYYFDYWGQGTSVTVSSGGGGSGGGGSGGGGSGGGGSDIVMTQTAPSVFVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTDFTLRISRVEAEDVGVYYCMQHLEYPVTFGAGT KVEIK Linker: SDPAHuman CD28STK spacer: KIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPTransmembrane derived from human IL-2Rβ: IPWLGHLLVGLSGAFGFIILVYLLIEndodomain derived from human IL-2Rβ:NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLVIllustrative construct with IL-7R alpha chain SEQ ID No. 46Signal sequence derived from human CD8a: MSLPVTALLLPLALLLHAAscFv aPSMA (J591 H/L) EVQLQQSGPELKKPGTSVRISCKTSGYTFTEYTIHWVKQSHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAAGWNFDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKWYWASTRHTGVPDRFTGSGSGTDFILTITNVQSEDLADYFCQQYNSYPLTFGAGTMLDLKR Linker SDPAHuman IgG1 Fc spacer (HCH2CH3pvaa):EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKTransmembrane derived from human CD28: FWVLVVVGGVLACYSLLVTVAFIIFWVEndodomain derived from TCRz:RRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT KDTYDALHMQ ALPPR2A peptide from Thosea asigna virus capsid protein: RAEGRGSLLTCGDVEENPGPSignal sequence derived from mouse kappa VIII: METDTLILWVLLLLVPGSTGscFv aPSA (5D5A5 H/L): QVQLQQSGAELAKPGASVKMSCKTSGYSFSSYWMHWVKQRPGQGLEWIGYINPSTGYTENNQKFKDKVTLTADKSSNTAYMQLNSLTSEDSAVYYCARSGRLYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIVLTQSPPSLAVSLGQRATISCRASESIDLYGFTFMHWYQQKPGQPPKILIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQTHEDPYTFGGGTKL EIK Linker: SDPAHuman CD8aSTK spacer: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDITransmembrane derived from human common gamma  chain:VVISVGSMGLIISLLCVYFWL Endodomain derived from human common gamma chain:ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET2A peptide from equine rhinitis A virus  polyprotein:QCTNYALLKLAGDVESNPGP Signal sequence derived from mouse kappa VIII:METDTLILWVLLLLVPGSTG scFv aPSA (5D3D11 H/L):QVQLQQSGPELVKPGASVKISCKVSGYAISSSWMNWVKQRPGQGLEWIGRIYPGDGDTKYNGKFKDKATLTVDKSSSTAYMQLSSLTSVDSAVYFCARDGYRYYFDYWGQGTSVTVSSGGGGSGGGGSGGGGSGGGGSDIVMTQTAPSVFVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTDFTLRISRVEAEDVGVYYCMQHLEYPVTFGAGT KVEIK Linker: SDPAHuman CD28STK spacer: KIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPTransmembrane derived from human IL-7Rα: PILLTISILSFFSVALLVILACVLWEndodomain derived from human IL-7Rα:KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ

Vector

The present invention also provides a vector, or kit of vectors, whichcomprises one or more nucleic acid sequence(s) encoding a one or moreCCR(s) according to the first aspect of the invention and optionally oneor more CAR(s). Such a vector may be used to introduce the nucleic acidsequence(s) into a host cell so that it expresses a CCR according to thefirst aspect of the invention.

The vector may, for example, be a plasmid or a viral vector, such as aretroviral vector or a lentiviral vector, or a transposon based vectoror synthetic mRNA.

The vector may be capable of transfecting or transducing a T cell or aNK cell.

Cell

The present invention provides a cell which comprises one or more CCR(s)of the invention and optionally one of more CAR(s).

The cell may comprise a nucleic acid or a vector of the presentinvention.

The cell may be a cytolytic immune cell such as a T cell or an NK cell.

T cells or T lymphocytes are a type of lymphocyte that play a centralrole in cell-mediated immunity. They can be distinguished from otherlymphocytes, such as B cells and natural killer cells (NK cells), by thepresence of a T-cell receptor (TCR) on the cell surface. There arevarious types of T cell, as summarised below.

Helper T helper cells (TH cells) assist other white blood cells inimmunologic processes, including maturation of B cells into plasma cellsand memory B cells, and activation of cytotoxic T cells and macrophages.TH cells express CD4 on their surface. TH cells become activated whenthey are presented with peptide antigens by MHC class II molecules onthe surface of antigen presenting cells (APCs). These cells candifferentiate into one of several subtypes, including TH1, TH2, TH3,TH17, Th9, or TFH, which secrete different cytokines to facilitatedifferent types of immune responses.

Cytolytic T cells (TC cells, or CTLs) destroy virally infected cells andtumor cells, and are also implicated in transplant rejection. CTLsexpress the CD8 at their surface. These cells recognize their targets bybinding to antigen associated with MHC class I, which is present on thesurface of all nucleated cells. Through IL-10, adenosine and othermolecules secreted by regulatory T cells, the CD8+ cells can beinactivated to an anergic state, which prevent autoimmune diseases suchas experimental autoimmune encephalomyelitis.

Memory T cells are a subset of antigen-specific T cells that persistlong-term after an infection has resolved. They quickly expand to largenumbers of effector T cells upon re-exposure to their cognate antigen,thus providing the immune system with “memory” against past infections.Memory T cells comprise three subtypes: central memory T cells (TCMcells) and two types of effector memory T cells (TEM cells and TEMRAcells). Memory cells may be either CD4+ or CD8+. Memory T cellstypically express the cell surface protein CD45RO.

Regulatory T cells (Treg cells), formerly known as suppressor T cells,are crucial for the maintenance of immunological tolerance. Their majorrole is to shut down T cell-mediated immunity toward the end of animmune reaction and to suppress auto-reactive T cells that escaped theprocess of negative selection in the thymus.

Two major classes of CD4+ Treg cells have been described—naturallyoccurring Treg cells and adaptive Treg cells.

Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+ Tregcells) arise in the thymus and have been linked to interactions betweendeveloping T cells with both myeloid (CD11c+) and plasmacytoid (CD123+)dendritic cells that have been activated with TSLP. Naturally occurringTreg cells can be distinguished from other T cells by the presence of anintracellular molecule called FoxP3. Mutations of the FOXP3 gene canprevent regulatory T cell development, causing the fatal autoimmunedisease IPEX.

Adaptive Treg cells (also known as Tr1 cells or Th3 cells) may originateduring a normal immune response.

The cell may be a Natural Killer cell (or NK cell). NK cells form partof the innate immune system. NK cells provide rapid responses to innatesignals from virally infected cells in an MHC independent manner

NK cells (belonging to the group of innate lymphoid cells) are definedas large granular lymphocytes (LGL) and constitute the third kind ofcells differentiated from the common lymphoid progenitor generating Band T lymphocytes. NK cells are known to differentiate and mature in thebone marrow, lymph node, spleen, tonsils and thymus where they thenenter into the circulation.

The CCR-expressing cells of the invention may be any of the cell typesmentioned above.

T or NK cells according to the first aspect of the invention may eitherbe created ex vivo either from a patient's own peripheral blood (1stparty), or in the setting of a haematopoietic stem cell transplant fromdonor peripheral blood (2nd party), or peripheral blood from anunconnected donor (3rd party).

Alternatively, T or NK cells according to the first aspect of theinvention may be derived from ex vivo differentiation of inducibleprogenitor cells or embryonic progenitor cells to T or NK cells.Alternatively, an immortalized T-cell line which retains its lyticfunction and could act as a therapeutic may be used.

In all these embodiments, CCR-expressing cells are generated byintroducing DNA or RNA coding for the or each CCR(s) by one of manymeans including transduction with a viral vector, transfection with DNAor RNA.

The cell of the invention may be an ex vivo T or NK cell from a subject.The T or NK cell may be from a peripheral blood mononuclear cell (PBMC)sample. T or NK cells may be activated and/or expanded prior to beingtransduced with nucleic acid encoding the molecules providing the CCRaccording to the first aspect of the invention, for example by treatmentwith an anti-CD3 monoclonal antibody.

The T or NK cell of the invention may be made by:

-   -   (i) isolation of a T or NK cell-containing sample from a subject        or other sources listed above; and    -   (ii) transduction or transfection of the T or NK cells with one        or more a nucleic acid sequence(s) encoding a CCR.

The T or NK cells may then by purified, for example, selected on thebasis of expression of the antigen-binding domain of the antigen-bindingpolypeptide.

Pharmaceutical Composition

The present invention also relates to a pharmaceutical compositioncontaining a plurality of cells according to the invention.

The pharmaceutical composition may additionally comprise apharmaceutically acceptable carrier, diluent or excipient. Thepharmaceutical composition may optionally comprise one or more furtherpharmaceutically active polypeptides and/or compounds. Such aformulation may, for example, be in a form suitable for intravenousinfusion.

Method of Treatment

The present invention provides a method for treating and/or preventing adisease which comprises the step of administering the cells of thepresent invention (for example in a pharmaceutical composition asdescribed above) to a subject.

A method for treating a disease relates to the therapeutic use of thecells of the present invention. Herein the cells may be administered toa subject having an existing disease or condition in order to lessen,reduce or improve at least one symptom associated with the diseaseand/or to slow down, reduce or block the progression of the disease.

The method for preventing a disease relates to the prophylactic use ofthe cells of the present invention. Herein such cells may beadministered to a subject who has not yet contracted the disease and/orwho is not showing any symptoms of the disease to prevent or impair thecause of the disease or to reduce or prevent development of at least onesymptom associated with the disease. The subject may have apredisposition for, or be thought to be at risk of developing, thedisease.

The method may involve the steps of:

-   -   (i) isolating a T or NK cell-containing sample;    -   (ii) transducing or transfecting such cells with a nucleic acid        sequence or vector provided by the present invention;    -   (iii) administering the cells from (ii) to a subject.

The T or NK cell-containing sample may be isolated from a subject orfrom other sources, for example as described above. The T or NK cellsmay be isolated from a subject's own peripheral blood (1st party), or inthe setting of a haematopoietic stem cell transplant from donorperipheral blood (2nd party), or peripheral blood from an unconnecteddonor (3rd party).

The present invention provides a CCR-expressing cell of the presentinvention for use in treating and/or preventing a disease.

The invention also relates to the use of a CCR-expressing cell of thepresent invention in the manufacture of a medicament for the treatmentand/or prevention of a disease.

The disease to be treated and/or prevented by the methods of the presentinvention may be a cancerous disease, such as bladder cancer, breastcancer, colon cancer, endometrial cancer, kidney cancer (renal cell),leukaemia, lung cancer, melanoma, non-Hodgkin lymphoma, pancreaticcancer, prostate cancer and thyroid cancer.

Where the ligand recognised by the CCR is PSA, the cancer may beprostate cancer.

The cells of the present invention may be capable of killing targetcells, such as cancer cells. The target cell may be characterised by thepresence of a tumour secreted ligand or chemokine ligand in the vicinityof the target cell. The target cell may be characterised by the presenceof a soluble ligand together with the expression of a tumour-associatedantigen (TAA) at the target cell surface.

The cells and pharmaceutical compositions of present invention may befor use in the treatment and/or prevention of the diseases describedabove.

The cells and pharmaceutical compositions of present invention may befor use in any of the methods described above.

Chimeric Transmembrane Protein

The present invention also provides a chimeric transmembrane proteincomprising a dimerization domain; and a cytokine receptor endodomain.

Dimerisation may occur spontaneously, in which case the chimerictransmembrane protein will be constitutively active. Alternatively,dimerization may occur only in the presence of a chemical inducer ofdimerization (CID) in which case the transmembrane protein only causescytokine-type signalling in the presence of the CID.

Suitable dimerization domains and CIDs are described in WO2015/150771,the contents of which are hereby incorporated by reference.

For example, one dimerization domain may comprise the rapamycin bindingdomain of FK-binding protein 12 (FKBP12), the other may comprise theFKBP12-Rapamycin Binding (FRB) domain of mTOR; and the CID may berapamycin or a derivative thereof.

One dimerization domain may comprise the FK506 (Tacrolimus) bindingdomain of FK-binding protein 12 (FKBP12) and the other dimerizationdomain may comprise the cyclosporin binding domain of cylcophilin A; andthe CID may be an FK506/cyclosporin fusion or a derivative thereof.

One dimerization domain may comprise an oestrogen-binding domain (EBD)and the other dimerization domain may comprise a streptavidin bindingdomain; and the CID may be an estrone/biotin fusion protein or aderivative thereof.

One dimerization domain may comprise a glucocorticoid-binding domain(GBD) and the other dimerization domain may comprise a dihydrofolatereductase (DHFR) binding domain; and the CID may be adexamethasone/methotrexate fusion protein or a derivative thereof.

One dimerization domain may comprise an 06-alkylguanine-DNAalkyltransferase (AGT) binding domain and the other dimerization domainmay comprise a dihydrofolate reductase (DHFR) binding domain; and theCID may be an 06-benzylguanine derivative/methotrexate fusion protein ora derivative thereof.

One dimerization domain may comprise a retinoic acid receptor domain andthe other dimerization domain may comprise an ecodysone receptor domain;and the CID may be RSL1 or a derivative thereof.

Where the dimerization domain spontaneously heterodimerizes, it may bebased on the dimerization domain of an antibody. In particular it maycomprise the dimerization portion of a heavy chain constant domain (CH)and a light chain constant domain (CL). The “dimerization portion” of aconstant domain is the part of the sequence which forms the inter-chaindisulphide bond.

The chimeric cytokine receptor may comprise the Fab portion of anantibody as exodomain, for example as illustrated schematically in FIG.5.

The chimeric transmembrane protein may comprise two polypeptides:

-   -   (i) a first polypeptide which comprises:        -   (a) a first dimerisation domain; and        -   (b) a first chain of the cytokine receptor endodomain; and    -   (ii) a second polypeptide which comprises:    -   (a) a second dimerization domain, which dimerises with the first        dimerization domain; and    -   (b) a second chain of the cytokine-receptor endodomain.

The sections above defining the cytokine receptor endodomain of thechimeric cytokine receptor also apply to the chimeric transmembraneprotein of the present invention.

The sections above relating to nucleic acids, vectors, kits, cells,pharmaceutical compositions and methods also apply to the chimerictransmembrane protein of the present invention.

The invention will now be further described by way of Examples, whichare meant to serve to assist one of ordinary skill in the art incarrying out the invention and are not intended in any way to limit thescope of the invention.

EXAMPLES Example 1—In Vitro Testing

T-cells are transduced with either a PSMA-specific CAR, or transducedwith a construct which co-expresses a PSMA-specific CAR with aPSA-specific CCR. T-cells are co-cultured with PSMA expressing targetcells which secrete or do not secrete PSA. This co-culture is conductedin the presence or absence of exogenous IL2. This co-culture isconducted at different effector to target ratios. This co-culture isrepeated serially with T-cells challenged with repeated target cells.Proliferation of T-cells and killing of target cells is determined. Inthis way, the contribution to proliferation and survival of T-cells theCCR makes can be measured. Further, the ability contribution to repeatedre-challenge the ability of serial

Example 2—In Vivo Testing

NSG mice are engrafted with a human prostate cancer cell line whichexpresses PSMA and secretes PSA and which expresses firefly Luciferase.T-cells are transduced with either a PSMA-specific CAR, or transducedwith a construct which co-expresses the PSMA-specific CAR with aPSA-specific CCR. T-cells are administered to the mice. The tumourburden can be serially measured using bioluminescent imaging and theresponse to CAR T-cells evaluated. Mice within each cohort can besacrificed at different time-points and tumour burden directly measuredby macroscopic measurements and by immunohistochemistry. Further,engraftment/expansion of T-cells at the tumour bed or within lymphoidtissues such as lymph nodes, spleen and bone-marrow measured by flowcytometry of said tissues.

Example 3—Creation of and Testing a Constitutively ActiveCytokine-Signalling Molecule

A constitutively active cytokine-signalling chimeric transmembraneprotein was produced by linking cytokine receptor endodomains to a “Fab”type exodomain (FIG. 5). This structure uses the natural dimerizationcomponents of antibodies, namely the dimerization domain from the heavyand light chain constant regions. The chimeric transmembrane protein hastwo chains; a first polypeptide which comprises the antibody light Kchain and the IL2 receptor common γ chain as endodomain; and a secondpolypeptide which comprises the antibody heavy chain CH1 and anendodomain which comprises either: the IL2 receptor β chain (giving aconstitutively active IL2-signalling molecule); or the IL7 receptor(giving a constitutively active IL7-signalling molecule). Theconstitutively active cytokine-signalling chimeric transmembraneproteins tested in this study included the scFv heavy and light chainvariable regions. These domains are not needed for dimerization tooccur. The signal is independent of antigen binding and the structurecould equally be “headless” (as shown in FIG. 5) or comprise anotherentity such as a protein tag.

Nucleic acid sequences encoding these two polypeptides were cloned inframe separated by a 2A-peptide encoding sequence.

CTLL-2 (ATCC® TIB-214TM) are murine cytotoxic T lymphocyte cells whichare dependent upon IL-2 for growth. In the absence of IL-2 the cellsundergo apoptosis. CTLL-2 cells were transduced with a vector expressingthe chimeric protein comprising an 12-receptor endodomain (Fab_IL2endo)or a vector expressing the chimeric protein comprising an 17 receptorendodomain (Fab_IL7endo) or left untransduced (WT). As a positivecontrol, cells of all three types were co-cultured with 100 U/ml murineIL2. Cell proliferation was assessed after 3 and 7 days of culture andthe results are shown in FIG. 6.

Untransduced CTLL2 cells, together with CTLL2 cells transduced witheither construct (Fab_IL2endo or Fab_IL7endo) proliferated in thepresence of 100 U/mL murine IL2 (FIG. 6, left-hand panel). However, inthe absence of exogenously added IL2, only cells transduced with theconstruct having an IL2R endodomain (Fab_IL2endo) survived andproliferated. This shows that the chimeric transmembrane receptorprovides the CTLL2 cells with the necessary IL2 signal.

Example 4—Generation and Testing of a Chimeric Cytokine Receptor AgainstPSA

A panel of chimeric cytokine receptors targeting PSA was developed usingscFvs derived from two antibodies which bind to different PSA epitopes:5D5A5 and 5D3D11. The crystal structure of PSA has been obtained in asandwich complex with these two (Stura et al (2011) as above).

Schematic diagrams illustrating some of the panel of CCRs is illustratedin FIG. 7.

The panel included the following constructs:A5-CD8stk-IL2Rg_D11-Hinge-IL2Rb: A CCR with an IL-2R endodomain havingA5 on the chain with common γ chain and D11 on the chain with the IL2R βchain;

D11-CD8stk-IL2Rg_A5-Hinge-IL2Rb: A CCR with an IL-2R endodomain havingD11 on the chain with common γ chain and A5 on the chain with IL2R βchain;

D11-CD8stk-RL_A5-Hinge-IL2Rb: A negative control construct which isequivalent D11-CD8stk-IL2Rg_A5-Hinge-IL2Rb, but in which the IL2Rγ chainis replaced by a rigid linker; D11-CD8stk-IL2Rg_A5-Hinge-IL7Ra: A CCRwith an IL-7R endodomain having D11 on the chain with common γ chain andA5 on the chain with IL7R α chain; and

D11-CD8stk-RL_A5-Hinge—IL7Ra: A negative control construct which isequivalent D11-CD8stk-IL2Rg_A5-Hinge-IL7Ra, but in which the IL2Rγ chainis replaced by a rigid linker;

CTLL2 cells were transduced with vectors expressing these constructs.Cells were cultured in the presence or absence of IL2 (the presence ofIL2 acting as a positive control) and the presence or absence of 5 ng/mLor 5 μg/mL PSA. CTLL2 cell proliferation was assessed after 3 and 7 daysand the results are shown in FIG. 8.

CTLL2 cells expressing a CCR with an IL7 endodomain did not supportCTLL2 cell survival and proliferation (FIG. 8, last two panels). Thepresence of murine IL-2 in these cells supported CTLL2 cell growth andproliferation at day 3, but by day 7 the majority of cells had undergoneapoptosis.

The anti-PSA chimeric cytokine receptors with an IL2R endodomainsupported CTLL2 cell proliferation in the absence of IL2 and thepresence of PSA at both 5 ng/ml and 5 μg/ml (FIG. 8, first panel), with5 μg/ml giving greater survival and proliferation, particularly at day7.

Both the anti-PSA chimeric cytokine receptors with an IL2R endodomain,i.e. A5-CD8stk-IL2Rg_D11-Hinge-IL2Rb and D11-CD8stk-IL2 Rg_A5-H inged L2Rb, indicating that the relative positioning of the two PSA-bindingdomains: 5D5A5 and 5D3D11, is not important for function.

Substitution of the common γ chain with a rigid linker abolished thecapacity of the CCR to support CTLL2 cell survival and proliferation(FIG. 8, third panel).

As another read-out for IL2 signalling, the phosphorylation of Y694 ofSTAT5 was investigated using phosphoflow.

CTLL2 cells were either untransduced (WT); transduced with a PSA CCRconstructs having an IL2R endodomain (D11-CD8STK-IL2Rg_A5-Hinge-IL2Rb);or transduced with an equivalent negative control construct in which theIL2Rγ chain is replaced with a rigid linker(D11-CD8STK-RLA5-Hinge-IL2Rb). The cells were incubated overnight in theabsence of exogenously added IL-2. The following day, the cells wereincubated with either Pervanadate at 500 μM (a positive control whichinhibits phosphatase and will lead to STAT5 phoshorylation) or 500 ng/mLPSA for 1 or 4 hours. After incubation the cells were fixed,permeabilised and analysed by flow cytometry.

The results are shown in FIG. 9. In the cells expressing the PSA CCR,the presence of PSA lead to increasing STAT5 phosphorylation with time(FIG. 9, central panel). No such increase in phosphorylation was seenwith untransduced CTLL2 cells, or with CTLL2 cells transduced with anequivalent construct in which the IL2Rγ chain is replaced with a rigidlinker (FIG. 9, right hand panel).

These results are consistent with the CTLL2 survival/proliferation datashown in FIG. 8 and demonstrate that a chimeric cytokine receptoragainst a soluble ligand (here, PSA) can be used to trigger cytokinesignalling in a T-cell.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

1-36. (canceled)
 37. A method for treating a subject for cancer whichcomprises the step of administering a pharmaceutical composition to thesubject, said pharmaceutical composition comprising a plurality ofcells, said cells comprising a chimeric transmembrane protein and achimeric antigen receptor (CAR), (A) wherein said chimeric transmembraneprotein comprises: (i) a first polypeptide which comprises (a) a firstdimerization domain comprising the dimerization portion of an antibodyheavy chain constant domain (C_(H)) and (b) a first chain of a cytokinereceptor endodomain; and (ii) a second polypeptide which comprises (a) asecond dimerization domain comprising the dimerization portion of anantibody light chain constant domain (C_(L)), which dimerizes with thefirst dimerization domain and (b) a second chain of thecytokine-receptor endodomain, wherein one of the first and second chainsof the cytokine receptor endodomain comprises an IL-2 receptor β-chainendodomain, an IL-7 receptor α-chain endodomain, or an IL-15 receptorα-chain endodomain, and wherein the other of the first and second chainsof the cytokine receptor endodomain comprises a γ-chain receptorendodomain that is common to the IL-2, IL-7, and IL-15 receptors; andwherein the first and second polypeptides spontaneously dimerize; and(B) wherein the CAR comprises an antigen-binding domain, a spacerdomain, a transmembrane domain, and an endodomain, wherein theantigen-binding domain specifically binds a tumour-associated antigen(TAA).
 38. The method according to claim 37, which comprises: (i)isolation of a cell-containing sample from a subject; (ii) transductionor transfection of T cells or NK cells from the sample with (a) anucleic acid construct or (b) a vector comprising the nucleic acidconstruct; said nucleic acid construct encoding the chimerictransmembrane protein and the CAR; and (iii) formulating the cells from(ii) to make the pharmaceutical composition.
 39. (canceled) 40.(canceled)
 41. The method according to claim 37, wherein: (i) the firstpolypeptide which comprises (a) a heavy chain constant domain (C_(H))and (b) a first chain of the cytokine receptor endodomain; and (ii) thesecond polypeptide which comprises (a) a light chain constant domain(C_(L)) and (b) a second chain of the cytokine-receptor endodomain. 42.The method according to claim 41, wherein the first polypeptidecomprises an antibody heavy chain variable domain (V_(H)) and a heavychain constant domain (C_(H)); and the second polypeptide comprises anantibody light chain variable domain (V_(L)) and a light chain constantdomain (C_(L)).
 43. The method according to claim 37, wherein the TAA isPSMA.
 44. The method according to claim 37, wherein the TAA is GD2. 45.A method for modulating a T cell or NK cell response in a subject, themethod comprising administering a pharmaceutical composition to thesubject, said pharmaceutical composition comprising a plurality of Tcells or NK cells, said cells comprising a chimeric transmembraneprotein, said chimeric transmembrane protein comprising: (i) a firstpolypeptide which comprises (a) a first dimerization domain comprisingthe dimerization portion of an antibody heavy chain constant domain(C_(H)) and (b) a first chain of a cytokine receptor endodomain; and(ii) a second polypeptide which comprises (a) a second dimerizationdomain comprising the dimerization portion of an antibody light chainconstant domain (C_(L)), which dimerizes with the first dimerizationdomain and (b) a second chain of the cytokine-receptor endodomain,wherein one of the first and second chains of the cytokine receptorendodomain comprises an IL-2 receptor β-chain endodomain, an IL-7receptor α-chain endodomain, or an IL-15 receptor α-chain endodomain,and wherein the other of the first and second chains of the cytokinereceptor endodomain comprises a γ-chain receptor endodomain that iscommon to the IL-2, IL-7, and IL-15 receptors; and wherein the first andsecond polypeptides spontaneously dimerize.
 46. The method according toclaim 45, wherein: (i) the first polypeptide which comprises (a) a heavychain constant domain (C_(H)) and (b) a first chain of the cytokinereceptor endodomain; and (ii) the second polypeptide which comprises (a)a light chain constant domain (C_(L)) and (b) a second chain of thecytokine-receptor endodomain.
 47. The method according to claim 45,wherein the first polypeptide comprises an antibody heavy chain variabledomain (V_(H)) and a heavy chain constant domain (C_(H)); and the secondpolypeptide comprises an antibody light chain variable domain (V_(L))and a light chain constant domain (C_(L)).